PROTEASE-CONTAINING MEMBRANES FOR RAPID, CONTROLLED ANTIBODY DIGESTION PRIOR TO MASS SPECTROMETRY ANALYSIS By Yongle Pang A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemistry-Doctor of Philosophy 2017 ABSTRACT PROTEASE-CONTAINING MEMBRANES FOR RAPID, CONTROLLED ANTIBODY DIGESTION PRIOR TO MASS SPECTROMETRY ANALYSIS By Yongle Pang Monoclonal antibodies are the fastest growing class of therapeutic drugs because of their high specificities to targeQt cells. Facile analysis of therapeutic mAbs and their post-translational modifications (PTMs) is essential for quality control, and mass spectrometry (MS) is the most powerful tool for antibody characterization. Conventional antibody characterization workflows contain an in-solution digestion step, which is labor-intensive and time-consuming. Proteasecontaining membranes are an attractive alternative platform for protein digestion because of their high local enzyme concentrations, short radial diffusion distances, rapid convection in pores, simple fabrication and low cost. Additionally, variation of protein residence time in the membrane gives control over the size of proteolytic peptides. This research focuses on developing workflows for monoclonal antibody characterization using functionalized porous membranes. Sequential adsorption of poly (styrene sulfonate) and pepsin in a porous nylon membrane forms a pepsin membrane reactor. Pepsin is inexpensive and catalyzes proteolysis in acidic solutions, which avoids the need to alkylate cysteine residues and limits antibody deamidation. Variation of the residence times (3 ms to 3 s) of antibody solutions in pepsin-containing membranes yields “bottom-up” (1-2 kDa) to “middle-down” (5-15 kDa) peptides in less than 10 min. These peptic peptides cover the entire sequences of Herceptin and a WatersTM antibody. Compared with the performance of bottom-up (in-solution tryptic digestion) and top-down (intact protein fragmentation) analysis of an antibody light chain, middle-down (in-membrane peptic digestion) analysis gives the highest bond cleavage (99%). In-membrane digestion also facilitates detection of PTMs such as oxidation, deamidation, N-terminal pyroglutamic acid formation and glycosylation. Recently developed protease-containing spin membranes provide an excellent platform for rapid, membrane-based protein digestion prior to ultrahigh-resolution Orbitrap MS analysis. Centrifugation of 100-200 ÂľL of pretreated protein solutions through the pepsin- or trypsincontaining membranes takes less than 1 min and gives nearly 100% coverage of the protein sequences in subsequent direct infusion MS analysis of digests of apomyoglobin and four commercial monoclonal antibodies (Herceptin, Avastin, Rituxan and Vectibix). MS analysis of peptic and tryptic peptides also reveals mAb PTMs such as N-terminal pyroglutamate formation, C-terminal Lysine clipping and glycosylation. Liquid chromatography coupled to tandem mass spectrometry analysis of tryptic spin digests and subsequent MaxQuant data searching show 100% sequence coverage of all four antibody light chains, and 75.1%-98.4% coverage of the heavy chains. Compared to in-solution tryptic digestion of mAbs, spin digestion yields higher sequence coverage and a larger number of unique peptides. In-membrane digestion also facilitates protein sequence comparison. Rapid peptic in-membrane digestion of two antibodies with direct infusion MS analysis accurately reveals the antibody modification site in less than 1 h. Overall, membrane-based protein digestion uses minimal sample preparation time and yields high peptide and sequence coverages for identification of protein PTMs. Copyright by YONGLE PANG 2017 I dedicate this dissertation to my parents, Chunming Pang and Jinlan Han, for their love and support. v ACKNOWLEDGEMENTS First I would like to thank my advisor Prof. Merlin Bruening for his continuous guidance and support over the past five years. He is very knowledgeable and helpful. His patience and trust encouraged me to overcome the obstacles along the way of pursuing my Ph.D. Merlin has excellent presentation skills, and he is always eager to share his experience of delivering complicated content to the audiences with different backgrounds. It is my honor to conduct my Ph.D. research with him, and I am very proud to be one of the alumni of the Bruening group. I want to give special thanks to Prof. Gavin Reid, who taught me the fundamental knowledge of mass spectrometry. I still remember the tips and tricks he taught me for mass spectrometry. Most of my MS data collection was conducted in Gavin’s lab. His enthusiasm for doing research inspired me to learn mass spectrometry step by step. I would also like to thank Prof. Daniel Jones, who gave me a lot of nice suggestions for doing research, getting prepared for conferences, and job hunting. I wish to thank Prof. Liangliang Sun for training me how to use data searching software. I also wish to thank Prof. Dana Spence for suggestions on this project. I would like to thank my collaborators, Prof. Donald Hunt and Dr. Weihan Wang for the antibody characterization project. Weihan was graduated from Bruening’s lab and gave me so much help in solving technical problems. I want to thank Dr. Gia Jokhadze from Clontech for manufacturing the spin columns, Dr. Mohammad Muhsin Chisti for providing the antibody samples, and Weijing Liu for the contribution to this dissertation. Without these collaborators’ help, I cannot finish the projects described here. vi I am also very grateful to all my fellow group members, including those who graduated before me and those who are currently working in the lab. In my early research stages, Dr. Yujing Tan and Dr. Jinlan Dong provided me so much help and brought me up to speed with my research. Yujing was very smart and hardworking. He is always a good example for me. Dr. Chao Cheng picked me up from the airport when I arrived in the US. I will always remember the the fun time we had together, and the sincere help from Bruening’s lab members. Lastly, I wish to express my deepest appreciation to my family and friends for their love and support. I am so fortunate to have my wonderful parents in my life. They always support me to chase my dream and do what I love to do. I would also like to thank my friends from Chinese Students and Scholar Association. The spectacular events we held and the wonderful time we had is a valuable memory for me. For my friends who have accompanied me in these years, I truly appreciate your continuous support and understanding. Our friendship will last no matter where we are. vii TABLE OF CONTENTS LIST OF TABLES ................................................................................................ xii LIST OF FIGURES ............................................................................................. xiv KEY TO ABBREVIATIONS.............................................................................. xix Chapter 1 . Introduction..........................................................................................1 1.1 Mass spectrometry for protein analysis ............................................................1 1.1.1 Ionization techniques ..................................................................................2 1.1.1.1 Matrix-assisted laser desorption/ionization ..........................................3 1.1.1.2 Electrospray ionization .........................................................................3 1.1.2 Mass analyzers ............................................................................................6 1.1.2.1 Time of flight mass analyzers ...............................................................7 1.1.2.2 Linear ion traps .....................................................................................8 1.1.2.3 Orbitrap analyzers .................................................................................8 1.1.3 Tandem mass spectrometry methods ........................................................11 1.1.3.1 Collision-induced activation/dissociation ..........................................12 1.1.3.2 Higher-energy C-trap dissociation .....................................................13 1.1.3.3. Electron Capture Dissociation and Electron Transfer Dissociation ..13 1.2 Monoclonal antibody analysis ........................................................................15 1.2.1 Monoclonal antibody market ....................................................................16 1.2.2 MS in mAb analysis..................................................................................20 1.2.2.1 Bottom-up mAb analysis ....................................................................22 1.2.2.2 Top-down approach for antibody analysis .........................................25 1.2.2.3 Middle-down approach for antibody analysis ....................................26 1.3 Protein-digestion methods ..............................................................................29 1.3.1 Enzymatic digestion ..................................................................................31 1.3.2 Approaches for rapid protein digestion ....................................................33 1.3.3 Immobilized enzyme reactors ...................................................................38 1.3.3.1 IMER supports ....................................................................................38 1.3.3.1.1 Monoliths ......................................................................................38 1.3.3.1.2 Capillaries .....................................................................................40 1.3.3.1.3 Magnetic beads .............................................................................42 1.3.3.1.4 Resins............................................................................................43 1.3.3.1.5 Microfluidic chips.........................................................................44 viii 1.3.3.2 In-membrane protein digestion...........................................................45 1.4 Outline of the dissertation ...............................................................................48 REFERENCES .....................................................................................................49 Chapter 2 . Pepsin-Containing Membranes for Controlled Monoclonal Antibody Digestion Prior to Mass Spectrometry Analysis ................................61 2.1 Introduction .....................................................................................................62 2.2 Experimental ...................................................................................................66 2.2.1 Materials ...................................................................................................66 2.2.2 Modification of Membranes with Pepsin .................................................67 2.2.3 mAb Reduction and Characterization.......................................................67 2.2.4 In-Membrane Digestion of Intact Antibody .............................................69 2.2.5 Digestion of the mAb Light and Heavy Chains .......................................69 2.2.5.1 In-membrane digestion .......................................................................69 2.2.5.2 In-solution digestion ...........................................................................69 2.2.6 “Top-down” Analysis of a mAb Light Chain ...........................................70 2.2.7 Mass Spectrometry and Data Analysis .....................................................70 2.3 Results and discussion ....................................................................................72 2.3.1 Protease-containing Membranes...............................................................72 2.3.2 mAb Reduction and Characterization.......................................................73 2.3.3 mAb Digestion in Membranes ..................................................................75 2.3.4 mAb Light-Chain Analysis using In-membrane or Tryptic In-solution Digestion ............................................................................................................91 2.3.5 Comparison of Light-chain Sequence Coverage Using “Middle-down”, “Bottom-up” and “Top-down” methods ............................................................92 2.3.6 Detecting PTMs on the light and heavy chains ........................................99 2.4 Conclusion ....................................................................................................103 2.5 Acknowledgement ........................................................................................104 REFERENCES ...................................................................................................105 Chapter 3 . Enzyme-Containing Spin Membranes for Rapid Protein Digestion ................................................................................................................................112 3.1 Introduction ...................................................................................................112 3.2 Experimental .................................................................................................115 3.2.1 Materials .................................................................................................115 3.2.2 Functionalized Membrane-Containing Spin Columns ...........................116 3.2.3 Apomyoglobin spin digestion with pepsin- and trypsin-containing membranes .......................................................................................................116 3.2.4 mAb spin digestion with pepsin- and trypsin-containing membranes ...117 3.2.4.1 In-membrane spin digestion of mAbs ..............................................117 ix 3.2.4.2 In-solution trypsin digestion of mAbs ..............................................118 3.2.5 Mass Spectrometry and Data Analysis ...................................................118 3.3 Results and discussion ..................................................................................120 3.3.1 Workflow for Digestion in Membrane-Containing Spin Columns ........120 3.3.2 Apomyoglobin spin digestion with pepsin- and trypsin-containing membranes .......................................................................................................121 3.3.3 mAb spin digestion with pepsin-containing membranes .......................126 3.3.4 mAb spin digestion with trypsin-containing membranes .......................160 3.3.5 LC/MS-MS analyses ...............................................................................182 3.4 Conclusions ...................................................................................................184 3.5 Acknowledgement ........................................................................................184 REFERENCES ...................................................................................................185 Chapter 4 . Membrane-base proteolytic digestion for protein sequence comparison ............................................................................................................191 4.1 Introduction ...................................................................................................191 4.2 Experimental .................................................................................................193 4.2.1 Materials .................................................................................................193 4.2.2 Manufacture of Pepsin-containing Membrane. ......................................194 4.2.3 Digestion of c13C6FR1_ZMapp and c13C6FR1_ZMapp +K antibodies with pepsin- containing membrane ..................................................................194 4.2.4 In-solution peptic digestion of c13C6FR1_ZMapp and c13C6FR1_ZMapp +K antibodies...................................................................195 4.2.5 In-solution tryptic digestion of c13C6FR1_ZMapp and c13C6FR1_ZMapp +K antibodies...................................................................195 4.2.6 Mass Spectrometry and Data Analysis ...................................................196 4.3 Results and discussion ..................................................................................196 4.3.1 Digestion of c13C6FR1_ZMapp and c13C6FR1_ZMapp +K antibodies in a pepsin-containing membrane ....................................................................197 4.3.2 In-solution peptic digestion of c13C6FR1_ZMapp and c13C6FR1_ZMapp +K antibodies...................................................................201 4.3.3 In-solution tryptic digestion of c13C6FR1_ZMapp and c13C6FR1_ZMapp +K antibodies...................................................................203 4.4. Conclusion ...................................................................................................204 4.5 Acknowledgement ........................................................................................205 REFERENCES ...................................................................................................206 Chapter 5 . Summary and future work .............................................................209 5.1 Research summary ........................................................................................209 5.2 Future work ...................................................................................................211 x 5.2.1 Limited proteolysis in protease-containing membranes to interrogate protein higher order structure ..........................................................................211 5.2.2 Polyclonal antibody digestion by protease-containing membranes .......214 5.2.3 De novo antibody sequencing .................................................................216 5.2.4 Glycosidase-containing membrane for haptoglobin deglycosylation ....221 5.3 Summary of future work ...............................................................................223 REFERENCES ...................................................................................................224 xi LIST OF TABLES Table 1.1. Marketed therapeutic monoclonal antibody products. ...........................17 Table 1.2. Common proteases and chemicals for catalysis of protein digestion. ....30 Table 1.3. Overview of Techniques for Accelerated Digestion. .............................37 Table 2.1. Light- and heavy-chain peptides identified from a 3-ms in-membrane digest of WIgG1. ......................................................................................................82 Table 2.2. Light- and heavy-chain peptides identified from a 3-s in-membrane digest of WIgG1. ......................................................................................................83 Table 2.3. WIgG1 peptides identified from an in-solution tryptic digest of the alkylated light chain. ................................................................................................93 Table 3.1. Apomyoglobin peptides identified from a spin-membrane (spun at 500 g) peptic digest. ..........................................................................................................125 Table 3.2. Apomyoglobin peptides identified from a spin-membrane (spun at 10,000 g) peptic digest. ..........................................................................................125 Table 3.3. Apomyoglobin peptides identified from a spin-membrane (spun at 500 g) tryptic digest. ..........................................................................................................126 Table 3.4. Light- and heavy-chain peptides identified from a spin-membrane (spun at 500 g) peptic digest of Herceptin. ......................................................................145 Table 3.5. Light- and heavy-chain peptides identified from a spin-membrane (spun at 500 g) peptic digest of Avastin. .........................................................................148 Table 3.6. Light- and heavy-chain peptides identified from a spin-membrane (spun at 500 g) peptic digest of Rituxan. .........................................................................151 Table 3.7. Light- and heavy-chain peptides identified from a spin-membrane (spun at 500 g) peptic digest of Vectibix. ........................................................................155 Table 3.8. Light- and heavy-chain peptides identified from a spin-membrane (spun xii at 500 g) tryptic digest of Herceptin. .....................................................................169 Table 3.9. Light- and heavy-chain peptides identified from a spin-membrane (spun at 500 g) tryptic digest of Avastin..........................................................................172 Table 3.10. Light- and heavy-chain peptides identified from a spin-membrane (spun at 500 g) tryptic digest of Rituxan. ..............................................................176 Table 3.11. Light- and heavy-chain peptides identified from a spin-membrane (spun at 500 g) tryptic digest of Vectibix. .............................................................178 Table 3.12. Antibody Sequence Coverages and Numbers of Unique Peptides Obtained From LC/MS-MS Analyses of Tryptic Spin and In-solution Digests. ..183 Table 4.1. MS signals that correspond to differences in that analysis of peptic inmembrane digestion of Z and ZK. .........................................................................201 xiii LIST OF FIGURES Figure 1.1. The ion-evaporation model and the charged residue model. ..................5 Figure 1.2. Part of the mass spectra of apomyoglobin peptic digests. ......................9 Figure 1.3. Schematic drawing of the LTQ Orbitrap Velos MS instrument with three new features compared with the old model LTQ Orbitrap. ............................11 Figure 1.4. Mechanisms of CID and ETD. ..............................................................14 Figure 1.5. Mechanisms of ADCC and CDC. .........................................................19 Figure 1.6. Structure of a monoclonal antibody. .....................................................20 Figure 1.7. A summary of different MS-based techniques for mAb characterization. ..................................................................................................................................22 Figure 1.8. Steps and intended effects in classical workflows for protein in-gel and in-solution digestion.................................................................................................34 Figure 1.9. A workflow of microwave-assisted protein digestion using immobilized trypsin on magnetic nanoparticles. ..........................................................................35 Figure 1.10. Scheme of the apparatus for real-time, on-line digestion of a protein separation. ................................................................................................................41 Figure 1.11. Experimental workflow for identifying proteins using an IMER. ......44 Figure 1.12. Setup for membrane reactor fabrication. .............................................46 Figure 1.13. Schematic workflow for membrane-based protein digestion. ............47 Figure 2.1.Workflow for controlled digestion and analysis of antibodies. .............65 Figure 2.2. mAb Reduction and Characterization ...................................................74 Figure 2.3. Part of the mass spectrum of an in-membrane digest (3-ms residence time) of reduced WIgG1 antibody. ..........................................................................75 xiv Figure 2.4. Part of the mass spectrum of a 3-ms, in-membrane digest of WIgG1. .77 Figure 2.5. Mass spectra of 3 different 3-ms, in-membrane digests of WIgG1. .....81 Figure 2.6. Part of the mass spectrum of a 3-ms, in-membrane digest of Trastuzumab. ............................................................................................................87 Figure 2.7. Deconvoluted ESI-Orbitrap mass spectra of the WIgG1 light chain digested with 3-ms (A) and 3-s (B) residence times in a pepsin-containing membrane. ................................................................................................................91 Figure 2.8. CID-MS/MS spectrum of the WIgG1 light-chain peptide L52-75, which covers the entire CDR-L2 region. .................................................................94 Figure 2.9 Summary of bond cleavage sites from CID, HCD and ETD-MS/MS of WIgG1 light-chain peptides obtained from a 30-ms digestion in pepsin-containing membranes. ..............................................................................................................95 Figure 2.10. Summary of the bond cleavage sites from CID and HCD-MS/MS of peptides obtained from tryptic, in-solution diges-tion of the WIgG1 Lc. ...............96 Figure 2.11 “Top-Down” HPLC MS/MS spectra of the WIgG1 light chain. The MS/MS parameters were 15-ms ETD (A, 13 scans merged), 5-ms ETD (B, 13 scans merged), and CID (C, 9 scans merged), respectively. ...................................97 Figure 2.12. Orbitrap FT MS/MS spectrum after Xtract deconvolution of the original MS/MS spectrum (Figure 2.11A) resulting from 15-ms ETD of the WIgG1 Lc..............................................................................................................................98 Figure 2.13. Cleavage sites in “top-down” analysis of the antibody light chain.....99 Figure 2.14. Part of the ESI-Orbitrap mass spectra of reduced WIgG1 light chains after digestion for 30-ms in pepsin-containing membranes. .................................100 Figure 2.15. Part of the CID-MS/MS spectra of light-chain amino acids L166-219 (top) and oxidized L166-219 (bottom), demonstrating the oxidation at methionine 180. .........................................................................................................................100 Figure 2.16. Part of the ESI-Orbitrap mass spectrum of a reduced WIgG1 heavy chain after in-membrane digestion with a residence time of 3 sec. .......................101 xv Figure 2.17. MS and CID-MS/MS spectra of the heavy-chain peptide H114-140 from a 3-s, in-membrane digestion. .......................................................................102 Figure 2.18. Part of the ESI-Orbitrap mass spectrum of a reduced WIgG1 heavy chain after digestion with a 3-s residence time in a pepsin-containing membrane. ................................................................................................................................103 Figure 3.1. Workflow for protein spin digestion and analysis. .............................120 Figure 3.2. Deconvoluted ESI-Orbitrap mass spectra of apomyoglobin peptic digests obtained through 500 g (top) and 10,000 g (bottom) spin digestion. ........122 Figure 3.3. Part of the mass spectrum of a tryptic spin digests (500 g) of apomyoglobin.........................................................................................................124 Figure 3.4. Sequence map of the peptides identified from infusion ESI-Orbitrap analysis of peptic digest of Avastin. ......................................................................128 Figure 3.5. Sequence map of the peptides identified from infusion ESI-Orbitrap analysis of peptic digest of Herceptin. ...................................................................129 Figure 3.6. Sequence map of the peptides identified from infusion ESI-Orbitrap analysis of peptic digest of Rituxan. ......................................................................130 Figure 3.7. Sequence map of the peptides identified from infusion ESI-Orbitrap analysis of peptic digest of Vectibix. .....................................................................131 Figure 3.8. Part of the mass spectrum of a peptic spin digest of Herceptin. .........133 Figure 3.9. Part of the mass spectrum of a peptic spin digest of Avastin..............136 Figure 3.10. Part of the mass spectrum of a peptic spin digest of Rituxan. ..........139 Figure 3.11. Part of the mass spectrum of a peptic spin digest of Vectibix. .........142 Figure 3.12. Mass spectra of 3 different spin-membrane digests of Avastin. .......159 Figure 3.13. Gel electrophoresis (SDS-PAGE) analysis of antibodies before and after digestion in a peptic spin column. .................................................................160 Figure 3.14. Part of the mass spectrum of a tryptic spin digest of Avastin. ..........162 xvi Figure 3.15. Sequence map of the peptides identified from infusion ESI-Orbitrap analysis of a tryptic digest of Herceptin. ...............................................................165 Figure 3.16. Sequence map of the peptides identified from infusion ESI-Orbitrap analysis of a tryptic digest of Avastin. ...................................................................166 Figure 3.17. Sequence map of the peptides identified from infusion ESI-Orbitrap analysis of a tryptic digest of Rituxan. ..................................................................167 Figure 3.18. Sequence map of the peptides identified from infusion ESI-Orbitrap analysis of a tryptic digest of Vectibix. .................................................................168 Figure 4.1. Parts of the sequences of 13C6FR1_ZMapp (Z) and c13C6FR1_ZMapp +K (ZK) antibodies. ...............................................................................................197 Figure 4.2. Workflow for comparison of two antibodies using pepsin-containing membranes for proteolysis. ....................................................................................198 Figure 4.3. Comparison of part of the mass spectra of in-membrane peptic digests of Z (top) and ZK (bottom). ...................................................................................199 Figure 4.4. Comparison of part of the MS/MS spectra of L107-115 from Z (top) and L107-116 from ZK (bottom). ..........................................................................200 Figure 4.5. Mass spectra of in-solution (top) and in-membrane (bottom) digests of Z. The maximum intensity is 1.02 x 106 in the top spectrum and 8.64 x 105 in the bottom spectrum. ....................................................................................................202 Figure 4.6. Part of the MS spectra of tryptic in-solution digests of Z (top) and ZK (bottom). This region shows different singly charged peaks. ...............................204 Figure 5.1. Direct infusion MS spectra of in-membrane tryptic digests (1.5-sec residence times) of Apo and Holo DS CRBPII dimers. ........................................213 Figure 5.2. Mass spectra of an in-membrane peptic digest (3-msec residence time) of a cocktail of four antibodies. .............................................................................215 Figure 5.3. Example of arranging peptides using relationships between their masses. ...................................................................................................................218 Figure 5.4. Comparison of mass spectra of haptoglobin tryptic peptides before (top) xvii and after (bottom) passing the tryptic digest through a glycosidase-containing membrane (3-sec residence time). .........................................................................222 xviii KEY TO ABBREVIATIONS ACN Acetonitrile ADCC Antibody-dependent cellular cytotoxicity ADCs Antibody-drug conjugates BLA Biologics License Application CAD Collision activation dissociation CDC Complement-dependent cytotoxicity CDRs Complementarity determining regions CESI-MS/MS Capillary electrophoresis-tandem mass spectrometry CH1, CH2, and CH3 heavy chain constant region 1, 2 and 3 CHAMPS Complete Homology-Assisted MS/MS Protein Sequencing CHO Chinese hamster ovary CI Chemical ionization CID Collision-induced dissociation CL light chain constant region CNBr Cyanogen bromide CRBPs Cellular retinol binding proteins CRM Charged residue model CS Chitosan xix DHB 2, 5-dihydroxy benzoic acid DTT Dithiothreitol ECD Electron capture dissociation EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride EI Electron ionization ESI Electrospray ionization ETD Electron transfer dissociation FA Formic acid FDA Food and Drug Administration FTICR Fourier transform ion cyclotron resonance HA Hyaluronic acid Hc Heavy chain HCD Higher-energy C-trap dissociation HCl Hydrochloric acid HDX Hydrogen deuterium exchange HOAc acetic acid IAM Iodoacetamide IdeS Streptococcus pyogenes IEM Ion evaporation model IgG Immunoglobulin G xx IMER Immobilized enzyme reactors INN International Nonproprietary Name IPA Isopropyl Alcohol Lc Light chain LC/MS Liquid chromatography coupled to mass spectrometry LIT Linear ion trap m/z Mass to charge MAA Marketing Authorization Application mAb Monoclonal antibody MALDI Matrix-assisted laser desorption/ionization MS Mass spectrometry MS/MS tandem mass spectrometry nanoESI Nanoelectrospray NEM N-ethylmaleimide NHS N-hydroxysuccinimide NTCB 2-nitro-5-thiocyanobenzoate OmpT Outer membrane protease T PAA Poly (acrylic acid) PNGaseF N-Glycosidase F PSS Polystyrene sulfonate xxi PTM Post-translational modifications Q Quadrupole RP-HPLC Reverse-phase liquid chromatography S/N Signal to noise SPS Shotgun protein sequencing TCEP Tris (2-carboxyethyl) phosphine hydrochloride TFA Trifluoroacetic acid TOF Time of flight UPLC-ESI-QTOF-MS Ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry UVPD ultraviolet photodissociation VH heavy chain variable region VL light chain variable region xxii Chapter 1 . Introduction This dissertation describes the fabrication and use of functionalized porous membranes for protein digestion prior to mass spectrometry (MS) analysis. More specifically, my research focuses on developing a workflow that uses pepsin-containing membranes to controllably digest monoclonal antibodies (mAbs) prior to MS analysis of their posttranslational modifications (PTMs) and sequences. Additionally, I investigate enzyme-containing spin membranes for rapid protein digestion and apply controlled membrane-based digestion to analyze protein sequences. To give a foundation for my work, this chapter provides important background on both MS and protein digestion. The introduction starts with an overview of MS-based protein analysis methods, summarizing ionization methods, mass analyzers and tandem mass spectrometry techniques that I used in my research. Then, I briefly review the mAb analysis methods (bottomup, top-down and middle-down strategies). The third part of the introduction describes protein digestion techniques, including traditional enzymes for protein digestion, techniques for accelerating protein digestion, and membrane-based protein digestion technology. Finally, this chapter outlines the subsequent chapters in this dissertation. 1.1 Mass spectrometry for protein analysis Over the past two decades, MS has advanced tremendously and become one of the most powerful analytical methods for characterizing biomolecules.1-4 MS exhibits high sensitivity, mass accuracy and resolution, along with high throughput and wide dynamic ranges.5 With the completion of the human genome project in 2003, researchers began to focus on the protein set produced or modified by an organism (proteomics), and the metabolites within cells, biofluids and tissues (metabolomics). MS plays a crucial role in proteomics studies because it can give 1 high-accuracy protein masses. Moreover, tandem mass spectrometry (MS/MS) can fragment the ions of interests to provide information on the protein sequence and PTMs.6 Additionally, current MS methods can quantify thousands of proteins from complex samples, which is crucial for proteomic studies.7 However, the extreme complexity of a proteome makes MS analysis challenging and continues to push the development of new MS instrumentation, robust analytical methodologies, laborsaving sample-preparation techniques, and user-friendly bioinformatics tools. Generally speaking, MS gives information on the masses of peptides and proteins. However, it does not directly determine the masses of molecules, but rather gives the mass to charge ratio (m/z) of ions. A MS experiment includes conversion of molecules to ions in the ionization source, separation of these ions (according to their m/z value) in the mass analyzer, detection of ions via electrical signals that depend on the ion abundance, and finally, processing the signals and producing a mass spectrum. This section briefly introduces ionization processes and mass analyzers that are fundamental to my work. 1.1.1 Ionization techniques The inventions of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) in the late 1980s greatly enhanced the scope of protein analysis using MS.8 Before that time, MS techniques such as electron ionization (EI) and chemical ionization (CI) could only ionize volatile analytes. Desorption/ionization techniques, including plasma desorption, fast atom bombardment or laser desorption, solve the ion production problem but suffer from poor signal to noise (S/N) ratios and show low intensities for compounds with molecular masses above 10,000. The development of two soft ionization techniques, MALDI 2 and ESI, reshaped the MS field. The method developers Koichi Tanaka and John Fenn received the Nobel Prize in chemistry in 2002 “for their development of soft desorption ionization methods for mass spectrometric analyses of biological macromolecules”. 1.1.1.1 Matrix-assisted laser desorption/ionization In 1988 Karas and Hillenkamp reported that matrix-ultraviolet laser desorption generates intact molecular ions of lysozyme, β-lactoglobulin A, porcine trypsin and albumin.9 The molar ratio of the organic molecule matrix to analyte in such experiments is around 5000:1. The excess matrix minimizes intramolecular interactions among the analyte molecules as well as damage to the analyte during the ionization process. Matrices such as 2, 5-dihydroxy benzoic acid (DHB) rapidly absorb the laser energy at a certain wavelength, normally 337 or 355 nm, to give an explosive breakdown of the analyte-matrix mixture and send these molecules into the gas phase. Initially, I analyzed proteins using a Thermo LTQ XL ion-trap mass spectrometer equipped with a vMALDI source. However, matrix molecules ionize along with the analyte, usually in the low mass range (<500 m/z), which makes characterization of small peptides challenging. 10 Also, the LTQ XL contains a linear ion trap, which has a maximum m/z cutoff of 4000. Considering that MALDI gives predominant singly charged peptide ions,11,12 and many of the peptides I analyze have molecular masses greater than 4000, I subsequently chose to use ESI as the primary ionization method. Nevertheless, MALDI has better tolerance towards salts and other contaminants than ESI.13 1.1.1.2 Electrospray ionization Also in 1988, John Fenn published his seminal work on using electrospray to form largemolecule ions with multiple charged states.14,15 In ESI, dissolved analyte enters a capillary, and 3 the electric field (~106 V/m) between the spray capillary and a counter electrode creates fine, highly charged droplets containing the analyte of interest. The dimensions of the droplets decrease with solvent evaporation, which increases the repulsion force between the charges in the droplet. The droplet continues to decrease in size until the repulsion force overcomes the surface tension, which results in droplet fission. At this point, the droplet releases a series of highly charged, tiny droplets. The Rayleigh equation, Eq (1-1), describes the maximum charge, 𝑄𝑅𝑦 , at which a droplet is stable. 𝑄𝑅𝑦 = 8𝜋(𝜀0 𝛾𝑅 3 )1/2 (1-1) In this equation, Îľ0 is the electrical permittivity; Îł is the surface tension of the solvent, and R is the radius of the droplet.16 The fission process continues until very small droplets form, and these are the precursors of the gas-phase ions. There are two mechanisms proposed for the formation of gas-phase ions from small droplets, the ion evaporation model (IEM) and the charged residue model (CRM).17 Figure 1.1 shows the two models. The IEM predicts that ion emission will occur when the droplet radius decreases to 10 nm, because at that point the field strength at the droplet surface is sufficiently large that Coulombic repulsion overcomes the energy required to increase the droplet surface area and expel an ion. Data for small ionic analytes support the IEM, but large analytes such as proteins likely form ions through the CRM.16 In the CRM, solvent evaporation results in an increase in the electric field strength, which is large enough (at the highest surface curvature) to form a Taylor Cone that emits small, highly charged droplets. Repetition of this process results in droplets containing only one analyte, and gas-phase ions form through evaporation and declustering of these final droplets. 4 Figure 1.1. The ion-evaporation model and the charged residue model. (A) The ion-evaporation model. An individual ion leaves the charged droplet in a solvated state. [Acronyms: kReaction, reaction rate constant; k, Boltzmann constant; T, temperature; h, Planck's constant, and R, ideal gas constant.] (B) The charged residue model. As solvent evaporates, smaller and smaller droplets form from a Taylor cone. Finally, droplets contain only one ion, and declustering or evaporation lead to the desolvated ion. [Acronyms: q, droplet charge at the Rayleigh instability limit; r, droplet radius; Îľ0, electric permittivity of the surrounding medium; Îł, surface tension, and σ, surface charge density.] Figure copied (with permission) from Matthias Wilm. Principles of electrospray ionization. Mol. & Cell Proteomics. 2011; Vol 10: M111.009407. Š the American Society for Biochemistry and Molecular Biology. ESI normally operates in three modes: direct infusion, nanospray infusion, or electrospray infusion coupled with liquid chromatography (LC/MS).10 Direct infusion ESI uses a syringe pump to introduce the sample to the ion source at a flow rate of several ÂľL/min. Nanospray employs a much lower flow rate with a pressurized, special nozzle.18 Commercially, the TriVersa NanoMate from Advion (Ithaca, NY) is a chip-based nanospray device. I conducted most of my work using the NanoMate, because a few ÂľL of sample can last for more than 30 min of 5 electrospray. The chip contains an array of nanoelectrospray (nanoESI) nozzles, and each nozzle is one-fifth the diameter of a hair. The most common mode for analyte introduction into the mass spectrometer, particular for mixtures, is LC-ESI-MS, where a protein or peptide mixture separates during flow through an LC column prior to ESI. This online sample analysis is advantageous for proteomics studies because offline fractionation causes significant sample loss. MS/MS of the peptides in the mass spectrometer provides sequence information, and modern software can match MS/MS data to peptide sequences in a database. As a well-established workflow, LC-ESI-MS/MS provides fast and robust qualitative and quantitative (with appropriate isotopic labeling or label-free methods) analysis of peptides. In contrast to MALDI, which predominantly forms singly charged ions, ESI gives multiply charged peptide or protein ions to enable characterization of peptides or proteins in the mass range of common mass spectrometers. The maximum charge state of a peptide typically corresponds well with the number of amino acids that can accept a proton (Lysine, Arginine, Histidine, and the N-terminus).10 Most of my data result from nanoESI-MS or LC-MS/MS analyses. 1.1.2 Mass analyzers After the ionization process, mass analyzers separate gas-phase ions based on their m/z values. The mass analyzer determines the resolution, accuracy, mass range, scan speed, ion transmission, and MS/MS capabilities of a mass spectrometer.19 The two main categories of mass analyzers include the scanning type in quadrupole (Q) and time of flight (TOF) instruments, and the trapping type in linear ion trap (LIT), Fourier transform ion cyclotron resonance (FTICR) and 6 Orbitrap instruments. State-of-the-art mass spectrometers often combine multiple mass analyzers to perform different types of experiments in one instrument. Also the arrangement of mass analyzers results in different instrument performance. In the following subsections, I briefly introduce the TOF, LIT and Orbitrap mass analyzers that I used to obtain MS and MS/MS data. 1.1.2.1 Time of flight mass analyzers Time of flight analyzers differentiate ions according to their velocities. An electric field first accelerates ions to convert their electrical potential energy into kinetic energy. Eq (1-2) describes the acceleration process 𝐸𝑘 = 𝑚𝑣 2 2 = 𝑞𝑉 = 𝑧𝑒𝑉 = 𝐸𝑒 (1-2) where Ek is the ion kinetic energy; m is the mass of the ion; v is the ion velocity; q is the total charge on the ion; z is the ion charge state; V is the electrical potential drop, and Ee is the electric potential energy. Eq (1-2) shows that the ion velocity depends on it mass-to-charge ratio. The TOF detector separates ions in time, as species with small m/z ratios reach the detector first. The first TOF analyzers were linear instruments that suffered from low resolution because of the range of kinetic energies gained by the same ions. Further development of TOF instruments with delayed pulsed extraction and reflectrons greatly enhanced resolution.10 TOF analyzers can couple with quadrupoles (QTOF) to give a robust hybrid instrument with the high resolution of the TOF analyzer and the MS/MS capability of the quadrupole. 7 1.1.2.2 Linear ion traps The linear ion trap is also known as a 2D ion trap. Thermo Scientific terms it an LTQ. In a LIT, the radial quadrupolar oscillating electric field and the axial DC electric field control ion trajectories.20 After the ions fly into the LIT, cooling occurs through collisions with inert gas, and the ions collect between the two ends of the trap (z axis). At the same time, radiofrequency potentials applied to the rods cause the ions to oscillate in the xy plane. Compared with a 3D ion trap (Paul ion trap), the LIT has ten times the ion-trapping capacity. The trapping efficiency increases from 5% for a 3D ion trap to 50% for LITs.19 Also, the larger internal space in the LIT solves the space-charge problem in the 3D ion trap. LIT analyzers have excellent MS/MS capabilities, and multiple-stage tandem mass spectrometry (MSn) is also possible, where a fragment ion is isolated for further fragmentation, which usually occurs through collisioninduced dissociation (CID).21 I will talk more fragmentation methods in later sections. Though the resolution of the LIT is not high, its relatively low price, robust operation, and easy maintenance, make it a very popular mass analyzer.22 1.1.2.3 Orbitrap analyzers Developed by Alexander Makarov, the Orbitrap is now a leading high resolution mass spectrometry analyzer in the MS field.23 FTICR instruments were the most common ultra-high resolution mass spectrometers before the invention of the Orbitrap. However, FTICR requires a superconducting magnet, which results in a high cost for operation and routine maintenance. Orbitrap analyzers can give a mass accuracy of sub parts per million,24 and have a maximum resolving power of 500,000 at m/z 200 (for Orbitrap Fusion Lumos Tribid mass spectrometers). Figure 1.2 illustrates the advantage of an LTQ-Orbitrap analyzer versus an LTQ analyzer in 8 resolving a 6+ charged ion. With the LTQ, one can determine a centroid mass of the ion, but not the charge state. The Orbitrap spectrum enables determination of the monoisotopic mass as well as the charge state (from the separation of the isotopic peaks). An accurate monoisotopic mass is vital for accurately identifying peptides without MS/MS data. Figure 1.2. Part of the mass spectra of apomyoglobin peptic digests. (A) Mass spectrum collected with an Orbitrap mass analyzer; (B) Mass spectrum collected with just the LTQ. The inserts (expanded regions above spectrum (A)) show the mass spectra collected with the Orbitrap (top) or just the LTQ (bottom insert) for a 6+ charged peptide. 9 The extraordinary performance of the Orbitrap results from its unique design. These analyzers contain two cup-shaped outer electrodes, and a spindle-like central electrode.19 Application of a voltage between the outer and central electrodes gives a linear electric field along the axis, which creates harmonic oscillations of ions. The radial electric field drags ions to the central electrode, and creates bent ion trajectories. When a packet of ions accumulated in the C-trap (a curved radiofrequency-only quadrupole ion trap between the LIT and orbitrap25) are injected into the Orbitrap, they begin rotating around the central electrode and oscillating between the outer electrodes. The outer electrodes detect the image currents created by the oscillations, to form the time-domain digital signals. The Fourier-transform of the time domain data yields frequency domain signals. As the frequency of the ion oscillation is inversely proportion to the square root of the m/z ratio of the ions, finally, a mass spectrum results. One of the limitations of the Orbitrap analyzer is that it cannot conduct MS/MS analysis, so normally it couples with other mass analyzers. Figure 1.3 shows the schematic of the LTQ Orbitrap Velos MS instrument that I used to acquire most of my infusion and LC-MS data. The Orbitrap family of mass spectrometers has grown rapidly in the past ten years. Normally, a new version of an Orbitrap instrument is introduced to the general community during the American Society of Mass Spectrometry annual conference. In the future, I expect to see Orbitrap analyzers with even higher acquisition speeds, resolving powers, mass accuracies and sensitivities. 10 Figure 1.3. Schematic drawing of the LTQ Orbitrap Velos MS instrument with three new features compared with the old model LTQ Orbitrap. “A, the stacked ring ion guide (S-Lens) increases the ion flux from the ESI source into the instrument by a factor 5–10; B, the dual linear ion trap design enables efficient trapping and activation in the high-pressure cell (left) and fast scanning and detection in the low pressure cell (right). C, the combo C-trap and HCD collision cell with an applied axial field with improved fragment ion extraction and trapping capabilities.”26 Figure taken (with permission) from Jesper V. Olsen, Jae C. Schwartz, Jens Griep-Raming, Michael L. Nielsen, Eugen Damoc, Eduard Denisov, Oliver Lange, Philip Remes, Dennis Taylor, Maurizio Splendore, Eloy R. Wouters, Michael Senko, Alexander Makarov, Matthias Mann, and Stevan Horning. A Dual Pressure Linear Ion Trap Orbitrap Instrument with Very High Sequencing Speed. Mol. Cell Proteomics. 2009; Vol 8:2759-2769. Š the American Society for Biochemistry and Molecular Biology. 1.1.3 Tandem mass spectrometry methods Proper selection of the ionization method and mass analyzer is vital for obtaining MS results that solve a specific problem. Determination of detailed protein or peptide sequence information 11 often requires MS/MS analysis of the target protein or peptide ions, and the amount of sequence data one obtains often depends on the fragmentation method. Below I discuss the fragmentation methods that I employ in this dissertation. 1.1.3.1 Collision-induced activation/dissociation Collision-induced dissociation (CID), also called collision activation dissociation (CAD), is the most common fragmentation method in MS/MS. It usually takes place in the collision cell of a mass spectrometer, the LIT or the Q2 of the triple quadrupole, for example.10 After applying a supplemental resonance excitation voltage to the x-axis, ions gain energy and collide with inert gas. A fraction of the ion translational energy transfers to internal energy, which brings the ion to an excited state. Subsequent unimolecular decomposition of the activated ion gives the products ions (b-, y-type ions). CID is an ergodic ion activation method, and redistribution of the internal energy results in fragmentation at the weakest bonds. Ions with a high charge state obtain more kinetic energy in a given electric field than low-charge-state ions, and thus have a higher probability of fragmenting.27 McLuckey et al. summarized the key experimental parameters in the CID of peptides and proteins ions.28 However, MS/MS spectra from a LIT suffer from low resolution and low mass accuracy. Also, fragment ions in the low m/z range are lost because of the low-mass cutoff that results from the radio frequency amplitude.29 Additionally, the fragmentation preference for the weakest bond makes locating labile protein PTMs, such as phosphorylation, challenging. Moreover, CID has limited value for intact protein fragmentation.19 CID includes an energy redistribution process, and for large ions such as proteins, energy redistribution goes through a large number of bonds. This limits the reaction rate for protein fragmentation. 12 1.1.3.2 Higher-energy C-trap dissociation The term higher-energy C-trap dissociation (HCD), coined by Olsen et al., specifically describes fragmentation in the octopole collision cell of the Orbitrap instrument.30 Different from CID MS/MS spectra of peptides, corresponding HCD spectra contains a2, b2 ion pairs and y1 and y2 ions, which helps the identification of reporter ions in the low m/z region. HCD is more efficient for intact protein fragmentation than CID.31 1.1.3.3. Electron Capture Dissociation and Electron Transfer Dissociation McLafferty and his coworkers developed electron capture dissociation (ECD) as a fragmentation method.32 A heated filament source outside the FTICR magnet produces low energy electrons (<0.2 eV), and a multiply charged positive ion captures an electron and forms a radical positive ion with reduced charge. An increase in ion internal energy because of dissociative recombination of an electron and the positive ion allows bond fission. ECD is a non-ergodic process, where no vibrational energy redistribution occurs.33 As such, ECD cleaves more bonds than CID, product ions come from single-bond cleavage, and labile PTMs and non-covalent bonds remain after ECD. I see many possible applications for protein fragmentation using ECD. However, the size of the electron beam is small compared to the volume of the ion trapping chamber, so the fragmentation efficiency is low. Moreover, ECD occurs mainly with the FTICR, not the LIT because the electric field in the LIT expels electrons, which limits their reaction with ions.19 To overcome this problem, the Hunt lab developed electron transfer dissociation (ETD). The mechanism of ETD involves electron transfer from a singly charged radical anion to multiply charged cations.34 This process releases 4-5.5 eV of energy, which triggers release of a hydrogen 13 radical. The fragmentation pathway is then the same as in ECD. ECD and ETD form c-, z-type ions. Figure 1.4 shows the production of b-, y-type ions and c-, z-type ions. Figure 1.4. Mechanisms of CID and ETD. (A) CID of a multiply protonated peptide for production of b- and y-type ions. (B) Reaction of a low-energy electron with a multiply protonated peptide produces c- and z-type ions. The picture is reprinted with permission from: John E. P. Syka, Joshua J. Coon, Melanie J. Schroeder, Jeffrey Shabanowitz, and Donald F. Hunt. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (26), 9528-33. Copyright (2004) Š National Academy of Sciences. 14 1.2 Monoclonal antibody analysis Antibodies are large glycoproteins produced by B lymphocytes in our immune system to identify and eliminate foreign objects such as viruses.35 In 1890, Behring and Kitasato published an article that showed that serum from an animal actively immunized against diphtheria toxin could neutralize even a fatal dose of the toxin in another animal.36 Since then, antibodies have been the subject of intense research and applications. In 1975, Kohler and Milstein developed the hybridoma technique,37 which makes the production of highly pure and specific mAbs possible in vivo38 and in vitro.39 Different from polyclonal antibodies, monoclonal antibodies are monospecific and homogeneous because they come from a single clone of immune cells. The general procedure for in vivo production of mAbs begins with immunization of an animal followed by isolation of B cells from the animal’s spleen. Cultivated myeloma cells are then fused with the isolated B cells to create hybridoma cells. Subsequently, the hybridomas that produce antibodies of desired specificities are selected and cloned to produce identical daughter clones.40 However, adverse human immune reactivity was an initial problem with mAbs derived from hybridoma cells. Most mAbs were initially developed from animals such as rabbits. When given to humans, these “foreign” bodies often evoked an immune reaction and were eliminated before reaching their targets. One way to solve this problem is to produce genetically engineered antibodies, such as chimeric antibodies and humanized antibodies.41 The three hypervariable loops, the complementarity determining regions (CDRs), on the light chain and heavy chain of an antibody determine the affinity and specificity of the antibody to a specific antigen. Thus, to build a recombinant humanized antibody, genes that encode the variable regions are fused with 15 genes that encode the remaining parts from a human antibody. This process removes most of the potentially immunogenic portions of the mAB, but its specificity for the intended therapeutic target does not change. 1.2.1 Monoclonal antibody market At the same time that advances in MS technology were reshaping protein analysis, monoclonal antibodies were becoming the fastest growing class of therapeutic drugs. In 1986, the US Food and Drug Administration (FDA) approved Muromonab-CD3 (Orthoclone OKT3), which became the first therapeutic antibody. Since then, the biotherapeutic market has continually increased, due in large part to the development of therapeutic mAbs, Fc-fusion proteins, antibody fragments, and antibody-drug conjugates (ADCs). More than 40 antibody-related drugs are commercially available, including the “big 5”: Rituximab (Rituxan, for Non-Hodgkin's lymphoma treatment), Infliximab (Remicade, for Crohn disease treatment), Trastuzumab (Herceptin, for breast cancer treatment), Adalimumab (Humira, for Rheumatoid arthritis treatment), and Bevacizumab (Avastin, for colorectal cancer treatment).42 Antibodies have a higher success rate (25-29%) from clinical phase I trials to approval than small molecules (11%).43 Their high specificity and low side effects are especially attractive. Table 1 shows marketed antibody products. In 2013, global sales for antibody products were estimated as $75 billion.44 16 Table 1.1. Marketed therapeutic monoclonal antibody products. Brand name (INN) Abthrax (raxibacumab) Actemra (tocilizumab) Adcetrisc (brentuximab vedotin) AlprolIXd (Factor IX Fc fusion protein) Arcalystf (rilonacept) Arzerra (ofatumumab) Avastin (bevacizumab) Benlysta (belimumab) Cimziag (certolizumab pegol) Cyramza (ramucirumab) Eloctateh (Factor VIII Fc fusion protein) Enbreli (etanercept) Entyvio (vedolizumab) Erbitux (cetuximab) j Eylea (aflibercept) Gazyva (obinutuzumab) Herceptin (trastuzumab) Humira (adalimumab) Ilaris (canakinumab) Original BLA/MAA Applicant Human Genome Sciences Roche Company Reporting US Sales GlaxoSmithKline Roche Company Reporting EU Sales N/Ab Roche Year of First Approval 2012 2009 2013 Global Sales ($M)a 23 1,119 Seattle Genetics Seattle Genetics Takeda Pharmaceutical Co. 2011 253 Biogen Idec Biogen Idec N/A 2014 NoMe N/A 2008 17 GlaxoSmithKline Genentech Human Genome Sciences Regeneron Pharmaceuticals GlaxoSmithKline Roche GlaxoSmithKline GlaxoSmithKline Roche GlaxoSmithKline 2009 2004 2011 117 6,748 228 UCB UCB UCB 2008 789 Eli Lilly and Co. Eli Lilly and Co. N/A 2014 NoMe Biogen Idec Biogen Idec N/A 2014 NoMe Immunex Takeda Pharmaceuticals U.S.A., Inc ImClone Systems Amgen Takeda Pharmaceutical Co. Bristol-Myers Squibb Regeneron Pharmaceuticals Roche Roche AbbVie Novartis Pharmaceuticals Pfizer 1998 8,325 Takeda Pharmaceutical Co. 2014 NoMe Merck KGaA Bayer Healthcare Pharmaceuticals Roche Roche AbbVie 2004 1,926 2011 1,851 2013 1998 2002 3 6,559 10,659 Novartis Pharmaceuticals 2009 119 Regeneron Pharmaceuticals Regeneron Pharmaceuticals Genentech Genentech Abbott Laboratories Novartis Pharmaceuticals kl Inflectra (infliximab [biosimilar]) Kadcylan (adotrastuzumab emtansine) Keytruda (pembrolizumab) Lemtrada (alemtuzumab) Hospira N/A Hospira 2013 <1m Genentech Roche Roche 2013 252 Merck & Co. Merck & Co. N/A 2014 NoMe Genzyme Therapeutics N/A Sanofi 2013 3 17 Table 1.1 (cont’d) Lucentiso (ranibizumab) Nplatep (romiplostim) Nulojixq (belatacept) Orenciar (abatacept) Perjeta(pertuzumab) Prolias (denosumab) Remicade (infliximab) Removabt (catumaxomab) Remsimak l (infliximab [biosimilar]) ReoProu (abciximab) Rituxan (rituximab) Simponi/ Simponi Aria (golimumab) Genentech Amgen Bristol-Myers Squibb Bristol-Myers Squibb Genentech Amgen Centocor Fresenius Biotech Roche Amgen Bristol-Myers Squibb Bristol-Myers Squibb Roche Amgen Johnson & Johnson N/A Novartis Pharmaceuticals Amgen Bristol-Myers Squibb Bristol-Myers Squibb Roche GlaxoSmithKline Merck & Co. NeoPharm Group 2006 2008 2011 2005 2012 2011 1998 2009 4,205 427 26 1,444 352 824 8,944 5 Celltrion N/A Celltrion 2013 <1m Centocor Genentech Lilly Roche N/A Roche 1994 1997 127 7,500 Centocor Ortho Biotech Johnson & Johnson Merck & Co. 2009 1,432 Novartis Pharmaceuticals 1998 30v Alexion Pharmaceuticals 2007 1,551 Johnson & Johnson Johnson & Johnson Abbvie Biogen Idec Amgen Amgen Novartis Bristol-Myers Squibb Sanofi 2009 2014 1998 2004 2006 2010 2003 2011 2012 1,504 NoMe 1,887 1,527 389 1,030 1,465 960 70 Spectrum Pharmaceuticals 2002 29 Simulect (basiliximab) Novartis Pharmaceuticals Soliris (eculizumab) Alexion Pharmaceuticals Stelara (ustekinumab) Sylvant (siltuximab) Synagis (palivizumab) Tysabri (natalizumab) Vectibix (panitumumab) Xgevas (denosumab) Xolair (omalizumab) Yervoy (ipilimumab) Zaltrapw (ziv-aflibercept) Zevalinx (ibritumomab tiuxetan) Janssen-Cilag International Janssen Biotech Abbott Laboratories Biogen Idec Amgen Amgen Genentech Bristol-Myers Squibb Sanofi Aventis IDEC Pharmaceuticals Novartis Pharmaceuticals Alexion Pharmaceuticals Johnson & Johnson Johnson & Johnson AstraZeneca Biogen Idec Amgen Amgen Roche Bristol-Myers Squibb Sanofi Spectrum Pharmaceuticals [Acronyms: INN, International Nonproprietary Name; BLA, Biologics License Application; MAA, Marketing Authorization Application]. a Sales information obtained from company annual reports and other publically available sources. b N/A denotes product not available in this region. c Antibody-Drug Conjugate. d Fc Fusion Protein, Fc-Factor IX. e Product approval in 2014; no sales in 2013. f Fc Fusion Protein, Fc-IL1R. g Fab Conjugate. h Fc Fusion Protein, Fc-Factor VIII. i Fc Fusion Protein, Fc-TNFR (p75). j Fc Fusion Protein, Fc-VEGFR (1,2). k Biosimilar Antibody, Remicade Originator. l Inflectra and Remsima are considered as two individual products. m Product approval in late 2013; no annual sales disclosed, bioTRAK estimate of global sales. n Antibody-Drug Conjugate. o Fab. p Fc Fusion Protein, Fc-TPO-R binding peptide. q Fc Fusion Protein, Fc-CTLA-4 with amino acid substitutions. r Fc Fusion Protein, Fc-CTLA-4. s Prolia and Xgeva are considered as two individual products even though they contain the same bulk monoclonal antibody. t Bispecific, Tri-functional Antibody. u Sales data not disclosed, small patient market, bioTRAKÂŽ estimate of global sales. v Fab, produced by papain digestion of full length monoclonal antibody. w Fc Fusion Protein, Fc-VEGFR. x Antibody Conjugate. Table is reprinted (with permission) from Dawn M Ecker, Susan Dana Jones, Howard L Levine. The therapeutic monoclonal antibody market. MAbs, 2015, 7 (1), 9-14. Copyright Š 2015 Taylor & Francis. 18 An antibody (in this dissertation antibody refers to immunoglobulin G, IgG) can potentially kill cancer cells by antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Figure 1.5 shows the mechanism for the antibody enhancing the effector function. ADCC is triggered by an interaction of the antibody Fc region and the FcÎł receptors (FcÎłRs) on immune effector cells, such as neutrophils, macrophages and natural killer cells. The tumor cell is killed by phagocytosis or lysis. In CDC, recruitment of the complement component C1q by IgG triggers a proteolytic cascade to activate the complement, which can lead to the formation of a membrane attack complex that kills the target cell by fracturing its cell membrane.41 Figure 1.5. Mechanisms of ADCC and CDC. This figure is reprinted with permission from Paul Carter. Improving the efficacy of antibody-based cancer therapies. Nat. Rev. Cancer, 1, 118-129. Copyright Š 2001, Rights Managed by Nature Publishing Group. 19 1.2.2 MS in mAb analysis mAbs, typically immunoglobulin G (IgG), have a molecular weight of approximately 150 kDa. As Figure 1.6 shows, they contain two identical light chains (Lc, ~25 kDa) and two identical heavy chains (Hc, ~50 kDa). The Lc contains a constant region (CL), and a variable region (VL), whereas the Hc has three constant regions (CH1, CH2, and CH3) and one variable region (VH). Inter- and intra-disulfide bonds connect the Lc and Hc. Figure 1.6. Structure of a monoclonal antibody. [Acronyms: VH, variable region of the heavy chain; CH1, CH2, and CH3, different constant regions of the heavy chain; CL, constant region of the light chain; VL, variable region of the light chain; Lc, light chain; Hc, heavy chain.] Manufacture of these large biomolecules is very different from synthesizing small molecules. Normally, therapeutic antibodies are expressed in mammalian host cell lines, including NS0 murine myeloma cells, PER.C6ÂŽ human cells, and Chinese hamster ovary (CHO) cells.45 PTMs such as glycosylation, N-terminal pyroglutamate formation, asparagine deamidation, C-terminal Lysine clipping, aspartic acid isomerization, oxidation, and degradation may result from 20 intracellular and extracellular processes during the expression, purification and storage. A recent review summarized the heterogeneity of monoclonal antibodies.46 Production of biosimilar antibodies (the same sequence) is a trend in the biopharmaceutical field; however, the inherent variability of antibody production makes exact copies of an effective antibody drug nearly impossible. The unintended byproducts may compromise product activity and stability. For example, oxidation of Met may cause an antibody conformational change, which affects binding to the antigen or Fc receptors.47 Deamidation of Asn on the variable region could results in loss of binding affinity.48 Thus, manufacturers must characterize the mAbs in detail after the production of each batch. Both the US FDA and the European Medicines Agency (EMA) require such control.49 The aforementioned heterogeneities of mAbs make their characterization challenging. Among the possible analytical techniques, MS is the most common and plays important roles throughout all the stages of mAb production due to its high accuracy and high-throughput capabilities. MS provides reliable information on clone selection, purification development, stability studies and comparability studies, and gives information related to primary sequences, PTMs, higher order structures and conformations of antibodies.50 Generally speaking, there are four strategies for antibody analysis: bottom-up, top-down, middle-up and middle-down approaches.51,52 Figure 1.7 summarizes different MS techniques for mAb characterization. 21 Figure 1.7. A summary of different MS-based techniques for mAb characterization. The figure is reprinted with permission from Zhongqi Zhang, Hai Pan, Xiaoyu Chen. Mass spectrometry for structural characterization of therapeutic antibodies. Mass Spectrom Rev., 2008, 28 (1), 147-176. Copyright Š 2008 Wiley Periodicals, Inc. 1.2.2.1 Bottom-up mAb analysis The bottom-up approach is the most widely used method for antibody characterization. In a typical workflow, after reduction and alkylation, the antibody light chain and heavy chain are digested by one or several enzymes in-gel or in-solution, and the proteolytic peptides are separated in reverse-phase liquid chromatography (RP-HPLC) for ESI-MS analysis. MS/MS analysis of the peptides also provides sequence information including the location of PTMs on the mAb. Although protein digestion can occur in different ways, most of the examples described below use in-solution digestion. I will further discuss protein digestion methods in later sections. 22 Many studies employed the bottom up procedure to investigate mAb variations and PTMs. Wang et al. used trypsin and Asp-N to digest huN901 antibody and separated the peptides with a C18 column prior to ESI-TOFMS analysis. 53 They identified N-terminal pyroglutamate formation, cleavage of C-terminal lysine, glycosylation, and deamidation on the huN901 antibody. Johnson et al. performed peptide mapping of an antibody after Asp-N digestion followed by RPHPLC/MS analysis, and revealed C-terminal Îą-amidation on the heavy chain.54 Ayoub et al. analyzed trypsin and GluC digests of cetuximab, and LC-MS/MS results suggested Ala213Glu substitution, and Cys214 missing in the light chain.55 Gahoual et al. conducted capillary electrophoresis-tandem mass spectrometry (CESI-MS/MS) analysis of Trastuzumab after trypsin in-solution digestion, and revealed the glycosylation profile.56 The Tsybin lab developed an “extended bottom-up” method using a novel enzyme, Sap 9, and achieved high sequence coverage of the light and heavy chains, with decreased introduction of artefacts during digestion.57 Wang et al. recently published their work on monitoring antibody PTMs by LC-MS with ultrafast tryptic digestion.58 Du et al. used 18 O-water as the solvent for mAb sample preparation followed by trypsin in-solution digestion.59 With this method they identified the deamidation artifacts introduced by sample preparation. Taylor Zhang and coworkers identified and characterized unpaired cysteines in a recombinant antibody.60 To locate the unpaired cysteines, they conducted trypsin digestion, and used Nethylmaleimide (NEM) to tag free thiols. Different from iodoacetamide, NEM can alkylate thiols at neutral or slightly acidic conditions. LC-MS analysis of tryptic peptides revealed free thiols at Cys-22 and Cys-96 in the variable region of the heavy chain. Xiang et al developed a two-step alkylation method for localization and quantitation of free thiols in mAbs. Briefly, the antibody was alkylated first with 12 C-iodoacetic acid, and subsequently was reduced and alkylated a 23 second time using 13 C-iodoacetic acid.61 Trypsin, Lys-C, chymotrypsin, Asp-N and Glu-C digests were analyzed by LC-MS. Peptides modified by 13 C-iodoacetic acid had a molecular weight 2 Da more than peptides modified by 12C-iodoacetic acid, which gives information about the free thiols. Hancock’s group used LC-MS with ETD to characterize mAb disulfide linkages.62 Tryptic in-gel digests (non-reducing gel) of mAbs were fragmented by CID and ETD. ETD preferentially fragments disulfide-linked peptides into two polypeptides, and CID (MS3) can further fragment these peptides to get more backbone cleavages. Using this method, they successfully identified the disulfide bonds scrambled under heat stress. Bottom-up methods are also widely used in hydrogen deuterium exchange (HDX) experiments for studying the antibody higher order structure.63 Pepsin routinely serves as the antibody protease after HDX because it is highly active under acidic conditions. Several papers summarized the protocols for using HDX.64,65 The bottom-up approach is the most powerful method for antibody analysis, but it still has limitations. Importantly, the procedure is usually time-consuming and labor-intensive. Artifact PTMs such as deamidation may occur during long incubations, and information about correlations between PTMs is lost because most PTMs reside on different peptides. Also, in LCMS/MS analysis, peptides larger than 4 kDa are hard to characterize by MS/MS, and peptides with two or three amino acids as well as hydrophilic peptides are easily lost during LC separation because of their poor retention on reverse-phase columns. CE may overcome some of these limitations of RP-LC, and we expect to see more research on antibody characterization using CE-MS.66 24 1.2.2.2 Top-down approach for antibody analysis Rapid improvements in ultrahigh resolution mass spectrometers have led to remarkable progress in the top-down approach for antibody characterization. This method determines the molecular mass of the intact protein and fragments the intact gas-phase protein ions without digestion.67 Top-down analysis can detect correlations between multiple PTMs, and sample preparation is simple compared with the bottom-up workflow. Fragmentation of the antibody can occur through CID, HCD, ECD, ETD, and ultraviolet photodissociation (UVPD), and the highresolution mass spectrometer is vital because the fragment ions are normally large. Zhang and Shah performed top-down analysis of the mAb variable regions via in-source fragmentation in a LTQ-Orbitrap instrument.68 In-source fragmentation occurs in the capillaryskimmer region and has the advantage that it can fragment all the charge states of the protein, which increases the sensitivity. They further conducted CID-MS/MS of specific fragment ions to obtain more sequence information. Bondarenko et al. conducted top-down HPLC/MS analysis of an IgG2 using an LTQ-Orbitrap.69 Using MagTran and ProMass software for ESI mass spectra deconvolution, they achieved a mass accuracy of the intact antibody within Âą2 Da (15 ppm). Insource CID of the intact IgG2 molecule showed a fragmentation pattern similar to their previous work. However, CID of an intact antibody yields limited sequence coverage. To increase the sequence coverage, Tsybin and coworkers conducted antibody analysis using a QTOF and ETD, because for large proteins ETD yields more fragmentation than CID.70 This instrument had a stated resolution up to 50,000 over a wide m/z range for intact protein, and up to 30,000-40,000 over a wide m/z range for fragment ions. The advantages of TOF versus Orbitrap and FTICR are larger dynamic range and single-ion counting. The TOF/ETD studies gave 21% sequence coverage for Murine MOPC 21 IgG, and 15% sequence coverage for human antiRhesus D IgG. 25 Tsybin’s group further conducted ETD of Humira on an Orbitrap Velos Pro. By averaging timedomain transients from different LC-MS experiments before FT signal processing, they obtained increased sensitivity, and higher sequence coverage (33%).71 Allan Marshall’s group recently used a 9.4 T FTICR to study an intact mAb.72 Simultaneous ECD of all the antibody charge states (42+ to 58+) yields more fragmentation (~34% sequence coverage) than ECD of one charge state (+51, ~25% sequence coverage). The limited sequence coverage for all these topdown approaches is mainly due to the highly structured and disulfide bond-protected areas. 1.2.2.3 Middle-down approach for antibody analysis The middle-down approach to protein characterization attempts to combine the strengths of bottom-up and top-down methods through analysis of large protein pieces (~3-20 kDa) obtained from limited digestion or reduction. Chemical or electrochemical reduction of an antibody forms free light chain and heavy chains. Some researchers use the term middle-up to describe the mass characterization of protein subunits and limited digests, while middle-down refers to MS/MS of the protein subunits or peptides from limited digestion. Tsybin used a new enzyme, Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS), to selectively cleave the antibody near the hinge region to give large peptides.73 MS/MS of the large peptides is easier than fragmentation of intact antibody. Also, LC separation of the large peptides yields greater resolution than separation of intact antibodies. Bondarenko et al. analyzed the reduced and alkylated antibody light chain and heavy chain using an LTQ-Orbitrap intrument. LC-MS/MS of the Lc and Hc yielded 53/213 bond cleavages for the Lc and 42/443 bond cleavages on the Hc.69 In-trap CID gave better sequence coverage than in-source CID 26 because of a reduction of noise by isolatinga 100 m/z range of precursor ions. In-source CID gave lower S/N, because ions from all the m/z range were fragmented. Jennifer Zhang and coworkers conducted limited proteolysis of an antibody using Lys-C with disulfide bond reduction to obtain ~25 kDa-sized Fab Hc, single Fc and Lc peptides.31 They separated the products of limited Lys-C digestion on a diphenyl HPLC column, and HCD analyses of the subunits showed 18/213 bond cleavages for deglycosylated single Fc, 25/214 for the Lc, and 29/226 of Hc Fab. Most of the fragmentation happened at the N termini of the subunits. Wang et al. used the middle-down approach to compare two anti-CD20 antibody drug products.74 IdeS digestion of two antibodies followed by tris (2-carboxyethyl) phosphine hydrochloride (TCEP) reduction yielded the Lc as well as the Fc and single chain Fab (Fd) on the HC. LC-MS analysis of the two digests revealed different PTM profiles on two products. They also suggested that a mass spectrometer with a resolution greater than 30,000 is suitable for the middle-down approach. Fornelli et al. described a similar protocol of using IdeS to first digest the antibody to Fc and F(ab’)2 , followed by TCEP reduction to form Fc, Fc/2 and Fd.73 They separated the subunits in a C4 column and fragmented them using ETD in an LTQ Orbitrap Elite instrument. Total-ion-current chromatograms showed clear separation of Fc/2, Lc and Fd. Isolation of the top 5 highly charged precursors on each subunit allowed efficient ETD. This procedure gave 67.6% sequence coverage for Fc/2, 68.5% for Lc and 58.6% for Fd. They also conducted IdeS digestion on an antibody mixture (three equimolar antibodies), and used LC to separate the subunits. Not surprisingly, the three Fc/2 subunits did not separate well because of over 90% sequence homology. 27 Simone Nicolardi and coworkers developed an online electrochemistry-assisted reduction of disulfide bonds workflow, and conducted fragmentation on the Lc using a 15 T FTICR.75 The authors tried different reducing conditions, but only achieved partial reduction of the antibody to obtain Lc, Hc, Lc+Hc, and Lc+2Hc pieces. CID of the Lc revealed two intrachain disulfide bonds. Yan et al. further tested the IdeS performance on different IgG subclasses (IgG1, IgG2, and IgG4), as well as an Fc fusion protein.76 Antibody subunits were separated on a C8 column, and interrogated with a Waters QTOF. They found that the cleavage sites were (PELL)G|G(P) for IgG1, (P.VA)G|G(P) for IgG2, (PEFL)G|G(P) for IgG4, and (PELL)G|G(P) for the Fc fusion protein. They concluded that this middle-down approach with IdeS proteolysis is convenient for IgG domain mapping. Deyun Wang et al. performed HCD middle-down MS/MS on the Lc and Hc of an antibody variant using a Q-Exactive Orbitrap.77 They separated the antibody and the variant by cationexchange chromatography, reduced the two fractions and separated the resulting mAb pieces on a C4 column. HCD fragmentation energy was optimized for the light chain precursor ion, because MS/MS of protein either by CID or ECD is charge-state-dependent. They finally achieved 46% sequence coverage of the Lc, and 20.3% coverage of the Hc. They further fragmented seven impurities, and concluded they contained site-specific modifications. Recently, Brodbelt’s group demonstrated a method for analyzing antibody subunits by 193 nm UVPD.78 They used IdeS to produce Lc, Fc/2 and Fd and analyzed these pieces using an Orbitrap Elite instrument modified with a 193 nm ArF excimer laser, which allows UVPD in the HCD collision cell. Tuning the pulse number and energy per pulse affords control of the fragmentation with the goal of maximizing sequence coverage of the three subunits. After combining data from 28 four independent UVPD experiments with different parameters, they obtained 80% sequence coverage for the Fc/2 and Lc, with lower than 70% coverage for the Fd peptide. Although the bottom-up method is still very common for mAb analysis, top-down and middledown approaches for antibody analysis have grown significantly in the past 5 years. With further improvements in MS technology, I expect to see more application of top-down and middle-down antibody analysis. 1.3 Protein-digestion methods Protein digestion does not occur solely in the laboratory, it happens in our body every second. Protein digestion begins with pepsinolysis in the stomach. The peptic peptides move to the duodenum for further digestion into amino acids using pancreatic enzymes such as trypsin, chymotrypsin and carboxypeptidase. The body then combines these amino acids to create new specialized proteins. Similarly, in the lab protein digestion helps us understand protein sequence and structure because smaller peptides are easier to characterize with modern analytical techniques. Catalysis of protein digestion can use enzymes or small molecules. Table 1.2 presents the commonly used proteases and chemicals for protein digestion. However, this section emphasizes enzymatic digestion. 29 Table 1.2. Common proteases and chemicals for catalysis of protein digestion.a protease Arg-C Asp-N Glu-C Lys-C Lys-N Trypsin Chymotrypsin Pepsin organism Clostridium histolyticum Pseudomonas fragi Staphylococcus aureus Lysobacter enzymogenes Lysobacter enzymogenes Bos taurus Bos taurus Sus scrofa Thermolysin Papain Pronase Bacillus thermoproteolyticus Carica papaya Streptomyces griseus specificity R’ ‘D E’b K’ ‘K81 K,R’ F,W,Y’ ‘F,L,W,Y’ ‘F,L’ ‘A,F,I,L,M,V R,K,D,H,G,Yb A,E,F,I,L,T,V,W,Y’ pH range 7.2–8.0b 7.0–8.0b 4.0–7.8b 8.5–8.8b 8.081 8.0b 7.0–9.0b 1.3 2 8.0c 6.0–7.0b 6.0–7.5b chemical CNBr HOAc FA HCl NTCB Hydroxylamine specificity M’ ‘D’79 D’ D’80 ‘C80 N–G pH range acidic acidic acidic 2.080 9 −1082 9.083 ‘ refers to cleavage at the C-terminus of the amino acid, and ’ refers to cleavage at the N-terminus of the amino acid. [Acronyms: formic acid (FA), hydrochloric acid (HCl), acetic acid (HOAc), cyanogen bromide (CNBr), 2-nitro-5-thiocyanobenzoate (NTCB)] a All data obtained from the Expasy bioinformatics resource portal (www.expasy.org), except those noted. b Roche Web site (www.roche-applied-science.com). c Sigma-Aldrich Web site (www.sigma-aldrich.com). Reprinted with permission from Linda Switzar, Martin Giera, Wilfried M. A. Niessen. Protein Digestion: An Overview of the Available Techniques and Recent Developments. J. Proteome Res., 2013, 12 (3), pp 1067–1077. Copyright (2013) American Chemical Society. 30 1.3.1 Enzymatic digestion As mentioned in the previous section, the bottom-up approach, which includes extensive digestion, is the gold standard for protein characterization.84 Trypsin is the most popular enzyme for bottom-up proteolysis because it is relatively cheap and can specifically cleave peptide bonds at the C-terminal side of Lysine (Lys) and Arginine (Arg), unless follow by Proline. Lys composes around 5.8% of the human proteome, and Arg shows a little lower abundance. The distribution of Lys and Arg in proteins results in tryptic peptides with an average length of 14 amino acids.85 Importantly, tryptic peptides contain at least two positions for protonation, and thus usually show at least two positive charges in ESI-MS. The small sizes of tryptic peptides allow effective LC separations, high MS ionization efficiency, and extensive CID-MS/MS fragmentation. Trypsin digestion normally occurs in-solution or in-gel. For the in-solution procedure, trypsinolysis typically follows reduction to break the disulfide bonds and alkylation to protect the thiol group from reforming disulfide bonds. After proper desalting and buffer exchange, in-solution digestion occurs at 37 °C for overnight. This protocol is ubiquitous. Trypsin is not always the ideal proteolytic enzyme. For instance, scientists conduct HDX experiments to study the protein higher-order structure. In this case, protein digestion has to take place in acidic conditions to prevent deuterium back exchange. Because trypsin is only active at neutral pH, it is not suitable for HDX studies. Pepsin is the most common enzyme for protein digestion in HDX because it is inexpensive and active in acidic conditions. Different from trypsin, however, pepsin is less specific and prefers to cleave proteins after hydrophobic amino acids.86 31 In addition to trypsin and pepsin, many other enzymes catalyze proteolysis. Although these enzymes are much more expensive than trypsin, their unique properties make them attractive for specific applications. Endoproteinase Lys-C, for example, is normally used in conjunction with trypsin to give complete protein digestion after Lys residues. In digestion of a yeast protein extract by trypsin alone, over 20% of the cleavage sites remain undigested, and the ratio of missed Lys to missed Arg is 5:1-6:1. Digestion using a Trypsin/Lys-C mixture gives a much lower percent of missed Lys. Lys-C can also catalyze protein digestion by itself, and the digests normally contain middle-down sized peptides. Similarly, Arg-C, Asp-N, and Glu-C can serve in middle-down approaches to protein digestion. Cong Wu et al. used outer membrane protease T (OmpT) for middle-down proteomics because this enzyme cleaves the bonds between two consecutive basic amino acids (Lys/Arg-Lys/Arg).87 The authors digested the 20-100 kDa proteins collected from the HeLa cell proteome and identified 3,697 unique peptides with an average peptide size of 6.3 kDa. OmpT peptides were also suitable for CID and ETD analysis. Huesgen and coworkers recently developed another new enzyme, LysargiNase, for protein digestion.88 Interestingly, LysargiNase mirrors trypsin in specificity, and has specific cleavages at the N-termini of basic residues. CID of LysargiNase results in a series of b-ions. When combined with the y-ion series from CID of trypsin digests, the peptide identification rate greatly improves. Lys-N has a similar activity but a lower specificity (only 71 % of the Lys-N digests have an N-terminal Lys). Although trypsin still dominates protein digestion because of its cost and specificity, applications that use other enzymes are increasing. 32 1.3.2 Approaches for rapid protein digestion Traditional workflows for bottom-up methods are usually time-consuming and labor-intensive. Figure 1.8 shows normal workflows for trypsin in-solution and in-gel digestion. Such complicated protocols are often bottlenecks for sample analysis. Thus, various techniques were developed to accelerate protein digestion.89 33 Figure 1.8. Steps and intended effects in classical workflows for protein in-gel and insolution digestion. The figure is reprinted with permission from J.L. Capelo, R. Carreira,M. Diniz, L. Fernandes,M. Galesio, C. Lodeiro, H.M. Santos, G. Vale. Overview on modern approaches to speed up protein identification workflows relying on enzymatic cleavage and mass spectrometry-based techniques. Anal. Chim. Acta, 2009, 650 (2), 151-9.. Copyright Š 2009 Elsevier B.V. All rights reserved. 34 High temperatures may decrease protein digestion time due to thermal protein denaturation.90 Partial unfolding of the protein increases its accessibility to the proteolytic enzyme. Although enzymes have an optimal working temperature, certain modifications can increase their thermal stability. For example, reductive methylation of trypsin increases its optimized working temperature to 50-60 °C.91 Microwave irradiation, which can take place in a simple microwave oven, can accelerate proteolysis and decrease digestion times to several minutes.92 Figure 1.9 shows a workflow of microwave-assisted protein. Figure 1.9. A workflow of microwave-assisted protein digestion using immobilized trypsin on magnetic nanoparticles. The figure is reprinted with permission from J.L. Capelo, R. Carreira,M. Diniz, L. Fernandes,M. Galesio, C. Lodeiro, H.M. Santos, G. Vale. Overview on modern approaches to speed up protein identification workflows relying on enzymatic cleavage and mass spectrometry-based techniques. Anal. Chim. Acta, 2009, 650 (2), 151-9. Copyright Š 2009 Elsevier B.V. All rights reserved. Ultrasonic energy also facilitates protein digestion. Different research groups used ultrasonic probes93, ultrasonic baths94 and sonoreactors95 to decrease protein digestion times to minutes. 35 High pressure also increases the rate of protein digestion due to pressure-induced protein denaturation. Tryptic digestion of bovine serum albumin can occur in 60 s at 35,000 psi.96 Gross and coworkers created an online, high pressure system for rapid digestion in HDX studies.97 Infrared energy provides another method for enhancing protein digestion efficiency. Excitation of the vibrations of N-H, C=O and C-N bonds of proteins and enzymes increases digestion rates.98 Table 1.3 gives a short summary of current techniques for accelerating protein digestion, including techniques that employ immobilized enzymes, which is the subject of the next section. 36 Table 1.3. Overview of Techniques for Accelerated Digestion. accelerated technique digestion time online compatibility specific applications High temperature Minutes (∟15) Not done, but possible Often applied to chemical digestion, not all proteases are thermostable Wide application area Membrane proteins (increased solubility), glycoproteins (decreased sterical hindrance) Microwave Minutes (≤15) Possible Compatible with proteases and chemical cleavage reagents Ultrasound Minutes (≤5) Not feasible Compatible with proteases and chemical cleavage reagents Wide application area High Pressure Seconds (<60) Yes Mostly done with enzymes HDX experiments (speed of online digestion-MS) Infrared Minutes (∟5) Not done Only advantageous for enzymes due to increased interaction with protein Wide application area Solvent Hours (≤5) Not done, possible but requires stop-flow strategy due to long digestion time Chemical digestion is often done in the presence of solvents, but some enzymes also tolerate relatively high percentages of organic solvent Membrane proteins (increased solubility) IMER* Minutes (≤20) Yes Compatible with each protease that retains activity when immobilized Wide application area Seconds (∟30) Yes Compatible with each protease that retains activity when immobilized Wide application area Seconds (5) Yes Compatible with each protease that retains activity when immobilized Wide application area Magnetic particle immobilized enzyme On-chip immobilized enzyme * IMER: Immobilized enzyme reactors. Reprinted with permission from Linda Switzar, Martin Giera, Wilfried M. A. Niessen. Protein Digestion: An Overview of the Available Techniques and Recent Developments. J. Proteome Res., 2013, 12 (3), pp 1067–1077. Copyright (2013) American Chemical Society. 37 1.3.3 Immobilized enzyme reactors Immobilized enzyme reactors (IMERs) are widely used for accelerating protein digestion. Immobilization of enzymes onto appropriate solid supports increases the enzyme stability under high temperature, over a broad pH range and in organic buffers.99-102 IMERs can serve in offline protein digestion as well as in online systems that reduce sample handling and achieve highthroughput analysis. A high local enzyme to substrate ratio yields rapid protein digestion, and immobilization limits autolysis of the enzyme. Specific IMERs were developed with a range of enzymes including trypsin, pepsin, chymotrypsin, Glu-C and Lys-C.100,103-105 Digestion in IMERs followed by MS analysis is becoming a common workflow for sample analysis. 1.3.3.1 IMER supports Enzyme immobilization can occur on a range of solid supports including monoliths, capillaries, magnetic particles, resins, microfluidic chips, and membranes. 1.3.3.1.1 Monoliths Monoliths are attractive IMER supports because of their low back pressure at high flow rates and fast mass transfer due to convection in small pores. Calleri et al. conducted online digestion of βlactoglobulin A and B in an epoxy-modified silica Chromolith SpeedRood support, and coupled it to a LC-MS/MS system for rapid protein digestion and identification.106 Digestion required less than 10 min. This group further studied the influence of the enzyme amount on the analytical performance.107 Krenkova et al. covalently immobilized L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin on a poly (glycidyl methacrylate-co-ethylene dimethacrylate) monolith in a fused silica capillary.108 Thirty-second online digestion with LC-MS analysis gave 38 80% sequence coverage of cytochrome c, which was similar to the result with 3 h of in-solution digestion. Masaru Kato et al. fabricated a trypsin IMER by coating a trypsin-containing gel (prepared by the sol-gel technique) on a porous silica monolith.109 Moreover, the silica monolith was developed to fit into a 96-well plate. The authors proposed to use this IMER for highthroughput protein analysis. Hanfa Zou’s group published work on coupling a monolithic capillary IMER with ÂľRPLCMS/MS for shotgun proteomics analysis.110 Digestion in the microreactor for several minutes was similar to 10 h of in-solution tryptic digestion. A one-minute digestion of 590 ng of yeast protein with IMER gave 1578 unique peptides. Ota and coworkers developed trypsin-containing monolithic silica within pipette tips, which is also known as MonoTipÂŽ Trypsin.111 Digestion occurred by pipetting the reduced and alkylated protein less than 20 times. Regine Schoenherr et al. presented a proof-of concept CE-pepsin microreactor-CE-MS/MS platform for protein analysis.112 CE separated intact protein in the first dimension, and after rapid protein digestion, CE peptide separation occurred in the second dimension. However, they obtained only 48% sequence coverage for cytochrome c and 22% for myoglobin. Nicoli et al. developed IMERs through immobilization of trypsin onto three monolith disks (CIMÂŽ epoxy disk, CIMÂŽ CDI disk, and CIMÂŽ EDA disk), and used them for online protein digestion and peptide mass fingerprinting.113 Results from 5 min of online digestion of five proteins with LC-ESI-MS/MS analysis was comparable with 20 h of in-solution digestion. Yukui Zhang’ group developed an organic-inorganic hybrid silica monolith trypsin IMER for protein digestion.114 The enzymatic activity of immobilized trypsin was about 6600 times greater than the activity of free trypsin in solution. A 150 s digestion of 20 Âľg of E. Coli protein using this 39 IMER lead to identification of 208 proteins, whereas 24 h in-solution trypsin digestion identified 176 proteins. Krenkova and coworkers immobilized trypsin and Lys-C on a poly(glycidyl methacrylate-coethylene dimethacrylate) monolith.115 They incorporated the IMER into an online system, and digested IgG in 6 min. The performance was similar to in-solution digestion for 24 h at 37 °C. Sinz’s group fabricated a capillary monolithic trypsin reactor and used it for online and offline trypsin digestion.116 They prepared a poly (glycidyl methacrylate-co-acrylamide-co-ethylene glycol dimethycrylate) monolith in a fused-silica capillary, and immobilized enzyme on the monolith through glutaraldehyde chemistry. They showed a 420-fold higher enzymatic activity of the immobilized trypsin compared to trypsin in solution. Sun et al. integrated a trypsin-IMER with CE-ESI-MS system for online protein digestion.66 The authors prepared an acrylamidebased monolith in a fused capillary, and tested the IMER performance by digestion of a sevenprotein mixture and a picogram quantity of RAW 264.7 cell lysate protein. Guihua Ruan and coworkers immobilized trypsin on a polymerized high internal phase emulsion monolith.117 Digestion of bovine serum albumin and cytochrome c in 10 min gave sequence coverages of 59% and 78%, respectively. The Dovichi group recently published their work on using a sulfonatesilica hybrid strong cation exchange monolith microreactor coupled to a polyacrylamide-coated capillary for online reduction, alkylation and digestion.118 They identified 3749 peptides after IMER digestion of 50 ng of Xenopus laevis zygote homogenate. 1.3.3.1.2 Capillaries Capillaries provide another attractive solid support for enzyme immobilization. Long and Wood immobilized pepsin onto a fused-silica capillary, and pulled it into a nanoESI emitter.103 They 40 coated the modified capillary and the nanoESI emitter with polyaniline to provide conductivity. Digestion of myoglobin gave comparable sequence coverage with in-solution pepsin digestion. Slysz et al. packed trypsin-modified beads in fused-silica capillaries and incorporated the reactor into an online digestion system.119 After protein separation in a capillary packed with C4 beads, the effluent was neutralized before passing through the IMER, and the resulting protein digest was acidified before injection into the mass spectrometer. Figure 1.10 presents a schematic workflow. The authors tested this integrated separation-digestion-MS system with a mixture of cytochrome c, myoglobin, carbonic anhydrase, and ovalbumin, and achieved sequence coverages of 74%, 100%, 53%, and 23%, respectively. The same group further developed the technology by conducting reversed-phase protein chromatography and rapid on-line tryptic digestion. Only 20 fmol of protein was needed for peptide mass fingerprinting.120 Figure 1.10. Scheme of the apparatus for real-time, on-line digestion of a protein separation. CapLc refers to a capillary LC system (Agilent 1000 Series, Waldbronn, Germany). Reprinted (with permission) from Gordon W. Slysz, David C. Schriemer. Blending Protein Separation and Peptide Analysis through Real-Time Proteolytic Digestion. Anal. Chem., 2005, 77 (6), pp 1572– 1579). Copyright (2005) American Chemical Society. Yamaguchi et al. prepared trypsin and chymotrypsin-IMERs using PTFE microtubes.121 The immobilization occurred through reaction of proteases with cross-linking agents (paraformaldehyde and glutaraldehyde). Protein digestion required 5 min without reduction and 41 alkylation. The sequence coverages of cytochrome c and BSA after digestion with the trypsinIMER were 47% and 12%, respectively. 1.3.3.1.3 Magnetic beads Magnetic particles are also popular substrates for enzyme immobilization. Yan Li and coworkers developed a trypsin-IMER with magnetic microspheres synthesized using a solvothermal reaction followed by coating with tetraethyl orthosilicate.122 Reaction of microspheres with aminopropyltriethoxysilane introduced amino groups, and activation with glutaraldehyde enabled covalent linking of trypsin to the magnetic silica microspheres through reaction with trypsin primary amines. After packing the magnetic microspheres into microchannels of a chip, digestion of cytochrome c for 5 min gave a sequence coverage comparable to that obtained after 12 h of in-solution digestion. Jeng et al. used trypsin-containing magnetic nanoparticles for rapid in situ protein digestion. Fe3O4 nanoparticles were prepared with –NH3+groups, and then trypsin was immobilized on the nanoparticles using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) chemistry.123 Protein digestion took place during incubation of the nanoparticles with the protein, and magnetic separation of the nanoparticles left protein digests in the solution. Digestion of lysozyme at 57 °C yielded 19 tryptic peptides with 98% sequence coverage. Sun and coworkers also used magnetic microspheres to prepare a trypsin-IMER. After activation of the he carboxylic acid-functionalized magnetic microspheres with N-hydroxysuccinimide (NHS) and EDC, trypsin amine groups reacted with succinimide groups on the magnetic microspheres to immobilize the enzyme. The authors tested the IMER with digestion of E. coli and the MCF7 cell line.66 Two to thirty min of trypsin-IMER digestion of E. coli enabled identification of 1300 42 proteins, whereas in-solution trypsin digestion lead to identification of 1400 proteins. For MCF7 cell line digestion, in-solution and IMER digestion also give similar numbers of protein identifications. 1.3.3.1.4 Resins Moore et al. developed a silica based IMER for online protein digestion.124 They immobilized trypsin onto ethylene bridged hybrid silica particles and packed the modified particles into a chromatographic column. This IMER can tolerate organic solvent, and was placed after the protein separation column to compare the performance of online/offline IMER digestion with insolution digestion. Protein/peptide elution was monitored by UV, and fractions were collected, recombined, and analyzed by a LC-MS system. With a 10-s volumetric residence time, online digestion of a yeast cell lysate identified 507 proteins, while in-solution digestion identified 490 proteins. Figure 1.11shows the detailed workflow. Freije and coworkers immobilized acetylated trypsin on Sepharose, Agarose (Pierce beads) and Poroszyme beads.125 They slurry-packed the beads with buffer into cartridges. Primary amino groups of Lysine and the N-terminus of trypsin were modified by acetic acid Nhydroxysuccinimide-ester to stabilize trypsin and enhance the cytochrome c digestion rate. Complete digestion occurred in a contact time of 4 s. 43 Figure 1.11. Experimental workflow for identifying proteins using an IMER. The figure is reprinted with permission from Stephanie Moore, Stephanie Hess, James Jorgenson. Characterization of an immobilized enzyme reactor for on-line protein digestion. J. Chromatogr. A, 2016, 1476, 1–8. . Copyright Š 2016 Elsevier B.V. All rights reserved. 1.3.3.1.5 Microfluidic chips Yun Liu and coworkers developed an IMER using layer-by-layer deposition of positively charged chitosan (CS), and negatively charged hyaluronic acid (HA) onto the surface of a poly (ethylene terephthalate) microfluidic chip.126 Finally, they soaked a chip modified with nine CS/HA bilayers in a solution containing trypsin. The value of Vmax per unit of trypsin (MichaelisMenton kinetics) was ∟600 mM/min Âľg, which is thousands of times faster than that in solution (0.2 mM/min Âľg). Liuni et al. made a microfluidic reactor by loading pepsin-agarose into a polymethyl methacrylate chip.127 After digestion with a residence time <4 s, MS sequence 44 coverages of myoglobin, bovine ubiquitin, and reduced bovine serum albumin were 99%, 64% and 66%, respectively. 1.3.3.2 In-membrane protein digestion Although many materials can support enzyme immobilization for protein digestion, microporous membranes present a unique platform because of their minimal thickness, large surface area-to volume ratio, low pressure drop, and convective flow through pores. Cooper et al. first used a membrane-based IMER for protein digestion. They immobilized trypsin in a poly(vinylidene difluoride) membrane using simple hydrophobic interactions and constructed a capillary fittingbased trypsin membrane reactor.128 Flow rates of 10 nL/min gave digests that showed complete sequence coverage of cytochrome c, and 20 nL/min flow rates through the capillary gave full sequence coverage of ovalbumin. The Bruening group developed several enzyme-containing membrane reactors for protein digestion. Xu and coworkers fabricated a trypsin-containing membrane by sequential deposition of polystyrene sulfonate (PSS) and trypsin in a nylon membrane.129 Figure 1.12 presents the membrane fabrication setup. 45 Figure 1.12. Setup for membrane reactor fabrication. (This figure was obtained from Zhefei Yang.) During flow of a solution through the membrane, PSS adsorbs to the membrane, presumably through hydrophobic interactions with the alkyl backbone of nylon, to give a negatively charged surface, even under acidic conditions. With a pI of ~10.5, trypsin has a positive charge in acidic solution, and can electrostatically adsorb to the negatively charged PSS on the surface. Layer-bylayer deposition on inexpensive nylon membranes gives a reactor with a local concentration of 10 mg trypsin per milliliter of membrane pores, which is 450 times higher than the trypsin concentration for in-solution digestion. The short Îźm radial diffusion distances in the membrane pores further contribute to facile digestion. Protein solutions were pushed through the membrane reactor with a syringe pump. Figure 1.13 shows a schematic digestion workflow. With such membranes, trypsin in-membrane digestion of Bovine Serum Albumin leads to higher sequence coverage (84%) than in-solution digestion (71%). 46 Figure 1.13. Schematic workflow for membrane-based protein digestion. (This figure was obtained from Zhefei Yang.) Tan et al. used a similar strategy to develop a pepsin-containing membrane. Moreover, the low thickness of the membrane (100 Îźm) affords control over membrane residence time at the millisecond level to limit digestion.130 Based on the work of previous group members, I developed a novel workflow for monoclonal antibody characterization using pepsin-containing membranes. By varying the residence times (from 3 ms to 3 s) of antibody solutions in the membrane, we obtained “bottom-up” (1-2 kDa) to “middle-down” (5-15 kDa) sized peptides, and these peptides cover the entire sequences of Herceptin and a Waters antibody.131 In a recent paper, Ning described using membranes attached to pipet tips for protein digestion. Proteolysis within pipet tips was more complete than digestion for 30 min in solution. Antibody digestion at the end of a pipet tip leads to 100% peptide coverage in MS analyses.132 From a high-throughput perspective, I further developed an enzyme-containing spin column for protein digestion. Digestion takes places during simple centrifugation of the protein solution through the spin membrane in 1 min or less, prior to analysis using direct infusion into an ultra-high resolution mass spectrometer or LC-MS/MS analysis. One-min peptic or tryptic spin-membrane digestion 47 yields nearly 100% sequence coverages of apomyoglobin and four commercialized monoclonal antibodies (Herceptin, Avastin, Rituxan and Vectibix). 1.4 Outline of the dissertation This dissertation contains four subsequent chapters. Chapter 2 describes antibody characterization using pepsin-containing membranes and MS analysis. Controlled in-membrane digestion generates bottom-up to middle-down sized peptides, and direct infusion MS and MS/MS analysis gives nearly 100% sequence coverage and identifies antibody PTMs. Chapter 3 presents a novel protein-digestion device, the enzyme-containing spin membrane. By inserting functionalized membranes into commercial spin columns, this new device provides protein digestion in less than 30 sec. Subsequently, chapter 4 describes the application of in-membrane digestion to determining the sequence differences between two antibodies. Finally, chapter 5 summarizes the aforementioned work and provides future research directions, such as, proteasecontaining membrane for protein higher order structure analysis, digestion of polyclonal antibodies, de novo antibody sequencing and fabrication of glycosidase-containing membranes. Specifically, the next four chapter titles are: Chapter 2: Pepsin-containing membranes for monoclonal antibody analysis Chapter 3: Enzyme-containing spin membranes for rapid protein digestion Chapter 4: Membrane-base proteolytic digestion for antibody sequence comparisons Chapter 5: Summary and future work 48 REFERENCES 49 REFERENCES (1) Deng, B.; Lento, C.; Wilson, D. J. Hydrogen deuterium exchange mass spectrometry in biopharmaceutical discovery and development - A review. Anal. Chim. Acta 2016, 940, 8. (2) Boughton, B. A.; Thinagaran, D.; Sarabia, D.; Bacic, A.; Roessner, U. Mass spectrometry imaging for plant biology: a review. Phytochem. Rev. 2016, 15, 445. (3) Baghdady, Y. Z.; Schug, K. A. Review of in situ derivatization techniques for enhanced bioanalysis using liquid chromatography with mass spectrometry. J. Sep. Sci. 2016, 39 (1), 102. (4) Senyuva, H. Z.; Gokmen, V.; Sarikaya, E. A. Future perspectives in Orbitrap-highresolution mass spectrometry in food analysis: a review. Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 2015, 32 (10), 1568. (5) Domon, B.; Aebersold, R. Mass spectrometry and protein analysis. Science 2006, 312 (5771), 212. (6) Larsen, M. R.; Trelle, M. B.; Thingholm, T. E.; Jensen, O. N. Analysis of posttranslational modifications of proteins by tandem mass spectrometry. Biotechniques 2006, 40 (6), 790. (7) Yates, J. R.; Ruse, C. I.; Nakorchevsky, A. Proteomics by mass spectrometry: approaches, advances, and applications. Annu. Rev. Biomed. Eng. 2009, 11, 49. (8) El-Aneed, A.; Cohen, A.; Banoub, J. Mass Spectrometry, Review of the Basics: Electrospray, MALDI, and Commonly Used Mass Analyzers. Appl. Spectrosc. Rev. 2009, 44 (3), 210. (9) Karas, M.; Hillenkamp, F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal. Chem. 1988, 60 (20), 2299. (10) Zhang, G.; Annan, R. S.; Carr, S. A.; Neubert, T. A. Overview of peptide and protein analysis by mass spectrometry. Curr. Protoc. Protein Sci. 2010, Chapter 16, Unit16 1. (11) Zenobi, R.; Knochenmuss, R. Ion formation in MALDI mass spectrometry. Mass Spectrom. Rev. 1998, 17 (5), 337. (12) Karas, M.; Gluckmann, M.; Schafer, J. Ionization in matrix-assisted laser desorption/ionization: singly charged molecular ions are the lucky survivors. J. Mass Spectrom. 2000, 35 (1), 1. Signor, L.; Boeri Erba, E. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometric analysis of intact proteins larger than 100 kDa. J. Vis. Exp. 2013, 79, 50635. (13) 50 (14) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Electrospray ionization for mass spectrometry of large biomolecules. Science 1989, 246 (4926), 64. (15) Meng, C. K.; Mann, M.; Fenn, J. B. Of Protons or Proteins - a Beams a Beam for a That (Burns,O.S.). Z. Phys. D Atom Mol. Cl. 1988, 10 (2-3), 361. (16) Kebarle, P. A brief overview of the present status of the mechanisms involved in electrospray mass spectrometry. J. Mass Spectrom. 2000, 35 (7), 804. (17) Wilm, M. Principles of electrospray ionization. Mol. Cell. Proteomics. 2011, DOI:10.1074/mcp.R111.009407 10.1074/mcp.R111.009407. (18) Wilm, M.; Mann, M. Analytical properties of the nanoelectrospray ion source. Anal. Chem. 1996, 68 (1), 1. (19) Hoffmann, E. d.; Stroobant, V. Mass spectrometry : principles and applications; 3rd ed ed.; J. Wiley: Chichester, West Sussex, England ; Hoboken, NJ, 2007. (20) Londry, F. A.; Hager, J. W. Mass selective axial ion ejection from a linear quadrupole ion trap. J. Am. Soc. Mass. Spectrom. 2003, 14 (10), 1130. (21) Schwartz, J. C.; Senko, M. W.; Syka, J. E. A two-dimensional quadrupole ion trap mass spectrometer. J. Am. Soc. Mass. Spectrom. 2002, 13 (6), 659. (22) Griffin, T. J.; Xie, H.; Bandhakavi, S.; Popko, J.; Mohan, A.; Carlis, J. V.; Higgins, L. iTRAQ reagent-based quantitative proteomic analysis on a linear ion trap mass spectrometer. J. Proteome. Res. 2007, 6 (11), 4200. (23) Makarov, A. Electrostatic axially harmonic orbital trapping: a high-performance technique of mass analysis. Anal. Chem. 2000, 72 (6), 1156. (24) Olsen, J. V.; de Godoy, L. M.; Li, G.; Macek, B.; Mortensen, P.; Pesch, R.; Makarov, A.; Lange, O.; Horning, S.; Mann, M. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol. Cell. Proteomics 2005, 4 (12), 2010. (25) Perry, R. H.; Cooks, R. G.; Noll, R. J. Orbitrap mass spectrometry: instrumentation, ion motion and applications. Mass Spectrom. Rev. 2008, 27 (6), 661. (26) Olsen, J. V.; Schwartz, J. C.; Griep-Raming, J.; Nielsen, M. L.; Damoc, E.; Denisov, E.; Lange, O.; Remes, P.; Taylor, D.; Splendore, M.et al. A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed. Mol. Cel.l Proteomics 2009, 8 (12), 2759. 51 (27) Watson, J. T.; Sparkman, O. D. Introduction to mass spectrometry : instrumentation, applications and strategies for data interpretation; 4th ed ed.; John Wiley & Sons: Chichester, England ; Hoboken, NJ, 2007. (28) Wells, J. M.; McLuckey, S. A. Collision-induced dissociation (CID) of peptides and proteins. Methods Enzymol. 2005, 402, 148. (29) Yang, Y. H.; Lee, K.; Jang, K. S.; Kim, Y. G.; Park, S. H.; Lee, C. S.; Kim, B. G. Low mass cutoff evasion with q(z) value optimization in ion trap. Anal. Biochem. 2009, 387 (1), 133. (30) Olsen, J. V.; Macek, B.; Lange, O.; Makarov, A.; Horning, S.; Mann, M. Higher-energy C-trap dissociation for peptide modification analysis. Nat. Methods 2007, 4 (9), 709. (31) Zhang, J.; Liu, H.; Katta, V. Structural characterization of intact antibodies by highresolution LTQ Orbitrap mass spectrometry. J. Mass Spectrom. 2010, 45 (1), 112. (32) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. Electron capture dissociation of multiply charged protein cations. A nonergodic process. J. Am. Chem. Soc. 1998, 120 (13), 3265. (33) Zubarev, R. A.; Haselmann, K. F.; Budnik, B.; Kjeldsen, F.; Jensen, F. Towards an understanding of the mechanism of electron-capture dissociation: a historical perspective and modern ideas. Eur. J. Mass Spectrom. 2002, 8 (5), 337. (34) Syka, J. E.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (26), 9528. (35) Chames, P.; Van Regenmortel, M.; Weiss, E.; Baty, D. Therapeutic antibodies: successes, limitations and hopes for the future. Br. J. Pharmacol. 2009, 157 (2), 220. (36) Llewelyn, M. B.; Hawkins, R. E.; Russell, S. J. Discovery of antibodies. BMJ 1992, 305 (6864), 1269. (37) Kohler, G.; Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975, 256 (5517), 495. (38) Insel, R. A. In vivo production of human hybridoma antibody to the Haemophilus influenzae B capsule in athymic nude mice. J. Infect. Dis. 1984, 150 (6), 959. (39) Pardue, R. L.; Brady, R. C.; Perry, G. W.; Dedman, J. R. Production of monoclonal antibodies against calmodulin by in vitro immunization of spleen cells. J. Cell Biol. 1983, 96 (4), 1149. (40) Breedveld, F. C. Therapeutic monoclonal antibodies. Lancet 2000, 355 (9205), 735. 52 (41) Carter, P. Improving the efficacy of antibody-based cancer therapies. Nat. Rev. Cancer 2001, 1 (2), 118. (42) Leavy, O. Therapeutic antibodies: past, present and future. Nat. Rev. Immunol. 2010, 10 (5), 297. (43) Reichert, J. M. Monoclonal Antibodies as Innovative Therapeutics. Curr. Pharm. Biotechno. 2008, 9 (6), 423. (44) Ecker, D. M.; Jones, S. D.; Levine, H. L. The therapeutic monoclonal antibody market. MAbs 2015, 7 (1), 9. (45) Li, F.; Vijayasankaran, N.; Shen, A. Y.; Kiss, R.; Amanullah, A. Cell culture processes for monoclonal antibody production. MAbs 2010, 2 (5), 466. (46) Liu, H.; Gaza-Bulseco, G.; Faldu, D.; Chumsae, C.; Sun, J. Heterogeneity of monoclonal antibodies. J. Pharm. Sci. 2008, 97 (7), 2426. (47) Zhang, A.; Hu, P.; MacGregor, P.; Xue, Y.; Fan, H.; Suchecki, P.; Olszewski, L.; Liu, A. Understanding the conformational impact of chemical modifications on monoclonal antibodies with diverse sequence variation using hydrogen/deuterium exchange mass spectrometry and structural modeling. Anal. Chem. 2014, 86 (7), 3468. (48) Du, Y.; Walsh, A.; Ehrick, R.; Xu, W.; May, K.; Liu, H. Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies. MAbs 2012, 4 (5), 578. (49) Beck, A.; Sanglier-Cianferani, S.; Van Dorsselaer, A. Biosimilar, biobetter, and next generation antibody characterization by mass spectrometry. Anal. Chem. 2012, 84 (11), 4637. (50) Beck, A.; Wagner-Rousset, E.; Ayoub, D.; Van Dorsselaer, A.; Sanglier-Cianferani, S. Characterization of therapeutic antibodies and related products. Anal. Chem. 2013, 85 (2), 715. (51) Zhang, Z.; Pan, H.; Chen, X. Mass spectrometry for structural characterization of therapeutic antibodies. Mass Spectrom. Rev. 2009, 28 (1), 147. (52) Zhang, H.; Cui, W.; Gross, M. L. Mass spectrometry for the biophysical characterization of therapeutic monoclonal antibodies. FEBS Lett. 2014, 588 (2), 308. Wang, L.; Amphlett, G.; Lambert, J. M.; Blattler, W.; Zhang, W. Structural characterization of a recombinant monoclonal antibody by electrospray time-of-flight mass spectrometry. Pharm. Res. 2005, 22 (8), 1338. (53) 53 (54) Johnson, K. A.; Paisley-Flango, K.; Tangarone, B. S.; Porter, T. J.; Rouse, J. C. Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain. Anal. Biochem. 2007, 360 (1), 75. (55) Ayoub, D.; Jabs, W.; Resemann, A.; Evers, W.; Evans, C.; Main, L.; Baessmann, C.; Wagner-Rousset, E.; Suckau, D.; Beck, A. Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middledown and bottom-up ESI and MALDI mass spectrometry techniques. MAbs 2013, 5 (5), 699. (56) Gahoual, R.; Busnel, J. M.; Beck, A.; Francois, Y. N.; Leize-Wagner, E. Full antibody primary structure and microvariant characterization in a single injection using transient isotachophoresis and sheathless capillary electrophoresis-tandem mass spectrometry. Anal. Chem. 2014, 86 (18), 9074. (57) Srzentic, K.; Fornelli, L.; Laskay, U. A.; Monod, M.; Beck, A.; Ayoub, D.; Tsybin, Y. O. Advantages of extended bottom-up proteomics using Sap9 for analysis of monoclonal antibodies. Anal. Chem. 2014, 86 (19), 9945. (58) Wang, Y.; Li, X.; Liu, Y. H.; Richardson, D.; Li, H.; Shameem, M.; Yang, X. Simultaneous monitoring of oxidation, deamidation, isomerization, and glycosylation of monoclonal antibodies by liquid chromatography-mass spectrometry method with ultrafast tryptic digestion. MAbs 2016, 8 (8), 1477. (59) Du, Y.; Wang, F.; May, K.; Xu, W.; Liu, H. Determination of deamidation artifacts introduced by sample preparation using 18O-labeling and tandem mass spectrometry analysis. Anal. Chem. 2012, 84 (15), 6355. (60) Zhang, T.; Zhang, J.; Hewitt, D.; Tran, B.; Gao, X.; Qiu, Z. J.; Tejada, M.; GazzanoSantoro, H.; Kao, Y. H. Identification and characterization of buried unpaired cysteines in a recombinant monoclonal IgG1 antibody. Anal. Chem. 2012, 84 (16), 7112. (61) Xiang, T.; Chumsae, C.; Liu, H. Localization and quantitation of free sulfhydryl in recombinant monoclonal antibodies by differential labeling with 12C and 13C iodoacetic acid and LC-MS analysis. Anal. Chem. 2009, 81 (19), 8101. (62) Wang, Y.; Lu, Q.; Wu, S. L.; Karger, B. L.; Hancock, W. S. Characterization and comparison of disulfide linkages and scrambling patterns in therapeutic monoclonal antibodies: using LC-MS with electron transfer dissociation. Anal. Chem. 2011, 83 (8), 3133. Pan, J.; Zhang, S.; Borchers, C. H. Comparative higher-order structure analysis of antibody biosimilars using combined bottom-up and top-down hydrogen-deuterium exchange mass spectrometry. Biochim. Biophys. Acta 2016, 1864 (12), 1801. (63) 54 (64) Li, J.; Rodnin, M. V.; Ladokhin, A. S.; Gross, M. L. Hydrogen-deuterium exchange and mass spectrometry reveal the pH-dependent conformational changes of diphtheria toxin T domain. Biochemistry 2014, 53 (43), 6849. (65) Wei, H.; Mo, J.; Tao, L.; Russell, R. J.; Tymiak, A. A.; Chen, G.; Iacob, R. E.; Engen, J. R. Hydrogen/deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications. Drug Discov. Today 2014, 19 (1), 95. (66) Sun, L.; Zhu, G.; Dovichi, N. J. Integrated capillary zone electrophoresis-electrospray ionization tandem mass spectrometry system with an immobilized trypsin microreactor for online digestion and analysis of picogram amounts of RAW 264.7 cell lysate. Anal. Chem. 2013, 85 (8), 4187. (67) Zhou, H.; Ning, Z.; Starr, A. E.; Abu-Farha, M.; Figeys, D. Advancements in top-down proteomics. Anal. Chem. 2012, 84 (2), 720. (68) Zhang, Z.; Shah, B. Characterization of variable regions of monoclonal antibodies by topdown mass spectrometry. Anal. Chem. 2007, 79 (15), 5723. (69) Bondarenko, P. V.; Second, T. P.; Zabrouskov, V.; Makarov, A. A.; Zhang, Z. Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-Orbitrap mass spectrometer. J. Am. Soc. Mass. Spectrom. 2009, 20 (8), 1415. (70) Tsybin, Y. O.; Fornelli, L.; Stoermer, C.; Luebeck, M.; Parra, J.; Nallet, S.; Wurm, F. M.; Hartmer, R. Structural analysis of intact monoclonal antibodies by electron transfer dissociation mass spectrometry. Anal. Chem. 2011, 83 (23), 8919. (71) Fornelli, L.; Damoc, E.; Thomas, P. M.; Kelleher, N. L.; Aizikov, K.; Denisov, E.; Makarov, A.; Tsybin, Y. O. Analysis of intact monoclonal antibody IgG1 by electron transfer dissociation Orbitrap FTMS. Mol. Cell. Proteomics 2012, 11 (12), 1758. (72) Mao, Y.; Valeja, S. G.; Rouse, J. C.; Hendrickson, C. L.; Marshall, A. G. Top-down structural analysis of an intact monoclonal antibody by electron capture dissociationFourier transform ion cyclotron resonance-mass spectrometry. Anal. Chem. 2013, 85 (9), 4239. (73) Fornelli, L.; Ayoub, D.; Aizikov, K.; Beck, A.; Tsybin, Y. O. Middle-down analysis of monoclonal antibodies with electron transfer dissociation orbitrap fourier transform mass spectrometry. Anal. Chem. 2014, 86 (6), 3005. Wang, B.; Gucinski, A. C.; Keire, D. A.; Buhse, L. F.; Boyne, M. T., 2nd. Structural comparison of two anti-CD20 monoclonal antibody drug products using middle-down mass spectrometry. Analyst 2013, 138 (10), 3058. (74) 55 (75) Nicolardi, S.; Deelder, A. M.; Palmblad, M.; van der Burgt, Y. E. Structural analysis of an intact monoclonal antibody by online electrochemical reduction of disulfide bonds and Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 2014, 86 (11), 5376. (76) An, Y.; Zhang, Y.; Mueller, H. M.; Shameem, M.; Chen, X. A new tool for monoclonal antibody analysis: application of IdeS proteolysis in IgG domain-specific characterization. MAbs 2014, 6 (4), 879. (77) Wang, D.; Wynne, C.; Gu, F.; Becker, C.; Zhao, J.; Mueller, H. M.; Li, H.; Shameem, M.; Liu, Y. H. Characterization of drug-product-related impurities and variants of a therapeutic monoclonal antibody by higher energy C-trap dissociation mass spectrometry. Anal. Chem. 2015, 87 (2), 914. (78) Cotham, V. C.; Brodbelt, J. S. Characterization of Therapeutic Monoclonal Antibodies at the Subunit-Level using Middle-Down 193 nm Ultraviolet Photodissociation. Anal. Chem. 2016, 88 (7), 4004. (79) Swatkoski, S.; Gutierrez, P.; Ginter, J.; Petrov, A.; Dinman, J. D.; Edwards, N.; Fenselau, C. Integration of residue-specific acid cleavage into proteomic workflows. Journal of Proteome Research 2007, 6 (11), 4525. (80) Smith, B. J. Chemical cleavage of polypeptides. Methods Mol. Biol. 2003, 211, 63. (81) Raijmakers, R.; Neerincx, P.; Mohammed, S.; Heck, A. J. Cleavage specificities of the brother and sister proteases Lys-C and Lys-N. Chem. Commun. 2010, 46 (46), 8827. (82) Tang, H. Y.; Speicher, D. W. Identification of alternative products and optimization of 2nitro-5-thiocyanatobenzoic acid cyanylation and cleavage at cysteine residues. Anal. Biochem. 2004, 334 (1), 48. (83) Crimmins, D. L.; Mische, S. M.; Denslow, N. D. Chemical cleavage of proteins in solution. Curr. Protoc. Protein Sci. 2005, Chapter 11, Unit 11 4. (84) Zhang, Y.; Fonslow, B. R.; Shan, B.; Baek, M. C.; Yates, J. R., 3rd. Protein analysis by shotgun/bottom-up proteomics. Chem. Rev. 2013, 113 (4), 2343. (85) Switzar, L.; Giera, M.; Niessen, W. M. Protein digestion: an overview of the available techniques and recent developments. J. Proteome. Res. 2013, 12 (3), 1067. (86) Hamuro, Y.; Coales, S. J.; Molnar, K. S.; Tuske, S. J.; Morrow, J. A. Specificity of immobilized porcine pepsin in H/D exchange compatible conditions. Rapid Commun. Mass Spectrom. 2008, 22 (7), 1041. 56 (87) Wu, C.; Tran, J. C.; Zamdborg, L.; Durbin, K. R.; Li, M.; Ahlf, D. R.; Early, B. P.; Thomas, P. M.; Sweedler, J. V.; Kelleher, N. L. A protease for 'middle-down' proteomics. Nat. Methods 2012, 9 (8), 822. (88) Huesgen, P. F.; Lange, P. F.; Rogers, L. D.; Solis, N.; Eckhard, U.; Kleifeld, O.; Goulas, T.; Gomis-Ruth, F. X.; Overall, C. M. LysargiNase mirrors trypsin for protein C-terminal and methylation-site identification. Nat. Methods 2015, 12 (1), 55. (89) Capelo, J. L.; Carreira, R.; Diniz, M.; Fernandes, L.; Galesio, M.; Lodeiro, C.; Santos, H. M.; Vale, G. Overview on modern approaches to speed up protein identification workflows relying on enzymatic cleavage and mass spectrometry-based techniques. Anal. Chim. Acta 2009, 650 (2), 151. (90) Park, Z. Y.; Russell, D. H. Thermal denaturation: a useful technique in peptide mass mapping. Anal. Chem. 2000, 72 (11), 2667. (91) Havlis, J.; Thomas, H.; Sebela, M.; Shevchenko, A. Fast-response proteomics by accelerated in-gel digestion of proteins. Anal. Chem. 2003, 75 (6), 1300. (92) Pramanik, B. N.; Mirza, U. A.; Ing, Y. H.; Liu, Y. H.; Bartner, P. L.; Weber, P. C.; Bose, A. K. Microwave-enhanced enzyme reaction for protein mapping by mass spectrometry: a new approach to protein digestion in minutes. Protein Sci. 2002, 11 (11), 2676. (93) Lopez-Ferrer, D.; Capelo, J. L.; Vazquez, J. Ultra fast trypsin digestion of proteins by high intensity focused ultrasound. J. Proteome. Res. 2005, 4 (5), 1569. (94) Shin, S.; Yang, H. J.; Kim, J.; Kim, J. Effects of temperature on ultrasound-assisted tryptic protein digestion. Anal. Biochem. 2011, 414 (1), 125. (95) Rial-Otero, R.; Carreira, R. J.; Cordeiro, F. M.; Moro, A. J.; Santos, H. M.; Vale, G.; Moura, I.; Capelo, J. L. Ultrasonic assisted protein enzymatic digestion for fast protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sonoreactor versus ultrasonic probe. J. Chromatogr. A 2007, 1166 (1-2), 101. (96) Lopez-Ferrer, D.; Petritis, K.; Hixson, K. K.; Heibeck, T. H.; Moore, R. J.; Belov, M. E.; Camp, D. G., 2nd; Smith, R. D. Application of pressurized solvents for ultrafast trypsin hydrolysis in proteomics: proteomics on the fly. J. Proteome. Res. 2008, 7 (8), 3276. (97) Jones, L. M.; Zhang, H.; Vidavsky, I.; Gross, M. L. Online, high-pressure digestion system for protein characterization by hydrogen/deuterium exchange and mass spectrometry. Anal. Chem. 2010, 82 (4), 1171. Wang, S.; Liu, T.; Zhang, L.; Chen, G. Efficient chymotryptic proteolysis enhanced by infrared radiation for peptide mapping. J. Proteome. Res. 2008, 7 (11), 5049. (98) 57 (99) Ma, J. F.; Zhang, L. H.; Liang, Z.; Shan, Y. C.; Zhang, Y. K. Immobilized enzyme reactors in proteomics. Trac-Trend Anal. Chem. 2011, 30 (5), 691. (100) Regnier, F. E.; Kim, J. Accelerating trypsin digestion: the immobilized enzyme reactor. Bioanalysis 2014, 6 (19), 2685. (101) Monzo, A.; Sperling, E.; Guttman, A. Proteolytic enzyme-immobilization techniques for MS-based protein analysis. Trac-Trend Anal. Chem. 2009, 28 (7), 854. (102) Ma, J. F.; Zhang, L. H.; Liang, Z.; Zhang, W. B.; Zhang, Y. K. Recent advances in immobilized enzymatic reactors and their applications in proteome analysis. Anal. Chim. Acta 2009, 632 (1), 1. (103) Long, Y.; Wood, T. D. Immobilized pepsin microreactor for rapid peptide mapping with nanoelectrospray ionization mass spectrometry. J. Am. Soc. Mass. Spectrom. 2015, 26 (1), 194. (104) Temporini, C.; Calleri, E.; Campese, D.; Cabrera, K.; Felix, G.; Massolini, G. Chymotrypsin immobilization on epoxy monolithic silica columns: development and characterization of a bioreactor for protein digestion. J. Sep. Sci. 2007, 30 (17), 3069. (105) Prikryl, P.; Ticha, M.; Kucerova, Z. Immobilized endoproteinase Glu-C to magnetic bead cellulose as a tool in proteomic analysis. J. Sep. Sci. 2013, 36 (12), 2043. (106) Calleri, E.; Temporini, C.; Perani, E.; De Palma, A.; Lubda, D.; Mellerio, G.; Sala, A.; Galliano, M.; Caccialanza, G.; Massolini, G. Trypsin-based monolithic bioreactor coupled on-line with LC/MS/MS system for protein digestion and variant identification in standard solutions and serum samples. J. Proteome. Res. 2005, 4 (2), 481. (107) Temporini, C.; Perani, E.; Mancini, F.; Bartolini, M.; Calleri, E.; Lubda, D.; Felix, G.; Andrisano, V.; Massolini, G. Optimization of a trypsin-bioreactor coupled with highperformance liquid chromatography-electrospray ionization tandem mass spectrometry for quality control of biotechnological drugs. J. Chromatogr. A 2006, 1120 (1-2), 121. (108) Krenkova, J.; Bilkova, Z.; Foret, F. Characterization of a monolithic immobilized trypsin microreactor with on-line coupling to ESI-MS. J. Sep. Sci. 2005, 28 (14), 1675. (109) Kato, M.; Inuzuka, K.; Sakai-Kato, K.; Toyo'oka, T. Monolithic bioreactor immobilizing trypsin for high-throughput analysis. Anal. Chem. 2005, 77 (6), 1813. (110) Feng, S.; Ye, M.; Jiang, X.; Jin, W.; Zou, H. Coupling the immobilized trypsin microreactor of monolithic capillary with muRPLC-MS/MS for shotgun proteome analysis. J. Proteome. Res. 2006, 5 (2), 422. (111) Ota, S.; Miyazaki, S.; Matsuoka, H.; Morisato, K.; Shintani, Y.; Nakanishi, K. Highthroughput protein digestion by trypsin-immobilized monolithic silica with pipette-tip formula. J. Biochem. Biophys. Methods 2007, 70 (1), 57. 58 (112) Schoenherr, R. M.; Ye, M.; Vannatta, M.; Dovichi, N. J. CE-microreactor-CE-MS/MS for protein analysis. Anal. Chem. 2007, 79 (6), 2230. (113) Nicoli, R.; Gaud, N.; Stella, C.; Rudaz, S.; Veuthey, J. L. Trypsin immobilization on three monolithic disks for on-line protein digestion. J. Pharm. Biomed. Anal. 2008, 48 (2), 398. (114) Ma, J.; Liang, Z.; Qiao, X.; Deng, Q.; Tao, D.; Zhang, L.; Zhang, Y. Organic-inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity. Anal. Chem. 2008, 80 (8), 2949. (115) Krenkova, J.; Lacher, N. A.; Svec, F. Highly efficient enzyme reactors containing trypsin and endoproteinase LysC immobilized on porous polymer monolith coupled to MS suitable for analysis of antibodies. Anal. Chem. 2009, 81 (5), 2004. (116) Spross, J.; Sinz, A. A capillary monolithic trypsin reactor for efficient protein digestion in online and offline coupling to ESI and MALDI mass spectrometry. Anal. Chem. 2010, 82 (4), 1434. (117) Ruan, G.; Wu, Z.; Huang, Y.; Wei, M.; Su, R.; Du, F. An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions. Biochem. Biophys. Res. Commun. 2016, 473 (1), 54. (118) Zhang, Z.; Sun, L.; Zhu, G.; Cox, O. F.; Huber, P. W.; Dovichi, N. J. Nearly 1000 Protein Identifications from 50 ng of Xenopus laevis Zygote Homogenate Using Online Sample Preparation on a Strong Cation Exchange Monolith Based Microreactor Coupled with Capillary Zone Electrophoresis. Anal. Chem. 2016, 88 (1), 877. (119) Slysz, G. W.; Schriemer, D. C. Blending protein separation and peptide analysis through real-time proteolytic digestion. Anal. Chem. 2005, 77 (6), 1572. (120) Slysz, G. W.; Lewis, D. F.; Schriemer, D. C. Detection and identification of subnanogram levels of protein in a nanoLC-trypsin-MS system. J. Proteome. Res. 2006, 5 (8), 1959. (121) Yamaguchi, H.; Miyazaki, M.; Honda, T.; Briones-Nagata, M. P.; Arima, K.; Maeda, H. Rapid and efficient proteolysis for proteomic analysis by protease-immobilized microreactor. Electrophoresis 2009, 30 (18), 3257. (122) Li, Y.; Yan, B.; Deng, C.; Yu, W.; Xu, X.; Yang, P.; Zhang, X. Efficient on-chip proteolysis system based on functionalized magnetic silica microspheres. Proteomics 2007, 7 (14), 2330. (123) Jeng, J.; Lin, M. F.; Cheng, F. Y.; Yeh, C. S.; Shiea, J. Using high-concentration trypsinimmobilized magnetic nanoparticles for rapid in situ protein digestion at elevated temperature. Rapid Commun. Mass Spectrom. 2007, 21 (18), 3060. 59 (124) Moore, S.; Hess, S.; Jorgenson, J. Characterization of an immobilized enzyme reactor for on-line protein digestion. J. Chromatogr. A 2016, 1476, 1. (125) Freije, J. R.; Mulder, P. P.; Werkman, W.; Rieux, L.; Niederlander, H. A.; Verpoorte, E.; Bischoff, R. Chemically modified, immobilized trypsin reactor with improved digestion efficiency. J. Proteome. Res. 2005, 4 (5), 1805. (126) Liu, Y.; Lu, H.; Zhong, W.; Song, P.; Kong, J.; Yang, P.; Girault, H. H.; Liu, B. Multilayer-assembled microchip for enzyme immobilization as reactor toward low-level protein identification. Anal. Chem. 2006, 78 (3), 801. (127) Liuni, P.; Rob, T.; Wilson, D. J. A microfluidic reactor for rapid, low-pressure proteolysis with on-chip electrospray ionization. Rapid Commun. Mass Spectrom. 2010, 24 (3), 315. (128) Cooper, J. W.; Chen, J.; Li, Y.; Lee, C. S. Membrane-based nanoscale proteolytic reactor enabling protein digestion, peptide separation, and protein identification using mass spectrometry. Anal. Chem. 2003, 75 (5), 1067. (129) Xu, F.; Wang, W. H.; Tan, Y. J.; Bruening, M. L. Facile trypsin immobilization in polymeric membranes for rapid, efficient protein digestion. Anal. Chem. 2010, 82 (24), 10045. (130) Tan, Y. J.; Wang, W. H.; Zheng, Y.; Dong, J.; Stefano, G.; Brandizzi, F.; Garavito, R. M.; Reid, G. E.; Bruening, M. L. Limited proteolysis via millisecond digestions in proteasemodified membranes. Anal. Chem. 2012, 84 (19), 8357. (131) Pang, Y.; Wang, W. H.; Reid, G. E.; Hunt, D. F.; Bruening, M. L. Pepsin-Containing Membranes for Controlled Monoclonal Antibody Digestion Prior to Mass Spectrometry Analysis. Anal. Chem. 2015, 87 (21), 10942. (132) Ning, W.; Bruening, M. L. Rapid Protein Digestion and Purification with Membranes Attached to Pipet Tips. Anal. Chem. 2015, 87 (24), 11984. 60 Chapter 2 . Pepsin-Containing Membranes for Controlled Monoclonal Antibody Digestion Prior to Mass Spectrometry Analysis (Part of this chapter was originally published in Analytical Chemistry. Reprinted with permission from Pang, Y., Wang, W. H., Reid, G. E., Hunt, D. F., Bruening, M. L. Anal. Chem. 2015, 87 (21), 10942-9. Copyright (2015) American Chemical Society. Work on “top-down” analysis was performed by Weihan Wang and Donald F. Hunt at the University of Virginia.) Monoclonal antibodies (mAbs) are the fastest growing class of therapeutic drugs because of their high specificities to target cells. Facile analysis of therapeutic mAbs and their post-translational modifications (PTMs) is essential for quality control, and mass spectrometry (MS) is the most powerful tool for antibody characterization. This study uses pepsin-containing nylon membranes as controlled proteolysis reactors for mAb digestion prior to ultra-high resolution Orbitrap MS analysis. Variation of the residence times (3 ms to 3 s) of antibody solutions in the membranes yields “bottom-up” (1-2 kDa) to “middle-down” (5-15 kDa) peptide sizes in less than 10 min. These peptides cover the entire sequences of Trastuzumab and a WatersTM antibody, and a proteolytic peptide comprised of 140 amino acids from the WatersTM antibody contains all three complementarity determining regions on the light chain. This work compares the performance of “bottom-up” (in-solution tryptic digestion), “top-down” (intact protein fragmentation) and “middle-down” (in-membrane digestion) analysis of an antibody light chain. Data from tandem MS show 99%, 55%, and 99% bond cleavage for “bottom-up”, “top-down”, and “middle-down” analyses, respectively. In-membrane digestion also facilitates detection of PTMs such as 61 oxidation, deamidation, N-terminal pyroglutamic acid formation and glycosylation. Compared to “bottom-up” and “top-down” approaches for antibody characterization, in-membrane digestion uses minimal sample preparation time, and this technique also yields high peptide and sequence coverage for identification of PTMs. 2.1 Introduction Monoclonal antibodies (mAbs) have emerged as an important class of biotherapeutic drugs with high selectivity and specificity,1-3 and the U.S. Food and Drug Administration (FDA) has approved more than 35 antibodies4 for treatment of diseases such as breast cancer,5 non-Hodgkin lymphoma,6 and colorectal cancer.7 According to the FDA’s “quality by design” policy, biotherapeutic materials such as mAbs must adhere to a consistent, predefined quality during manufacturing.8 Facile mAb characterization, especially in the complementarity determining regions (CDRs), is vital for quality control, not only because these proteins are vulnerable to chemical modifications during expression, purification and long-term storage, but also because they have natural heterogeneities.9-11 Common PTMs on mAbs include methionine oxidation, asparagine deamidation, asparagine glycosylation in the heavy-chain constant region 2 (CH2), and heavy chain C-terminal processing.12-21 MS is the most powerful tool for antibody characterization because of its high resolution and mass accuracy within a wide dynamic range. Current MS-based strategies for antibody characterization employ “top-down”,22-25 “bottom-up”26-35 and “middle-down”36-40 approaches with ultra-high resolution time-of-flight (TOF),24,27,29,30,33-35,39 Fourier transform ion cyclotron resonance25,41 and Orbitrap22,23,26,36-38,40 mass spectrometers. “Top-down” methods introduce the intact antibody into the mass spectrometer through liquid chromatography (LC) or direct infusion. 62 Determination of the intact protein mass and subsequent gas-phase fragmentation via collisioninduced dissociation (CID),22,23 higher-energy collision dissociation (HCD), electron capture dissociation (ECD),25 or/and electron transfer dissociation (ETD)24 give an overview of the major PTMs with minimal sample manipulation time. Unfortunately PTMs with small mass changes, e.g. deamidation (+1 Da), cannot be detected, and sequence coverage for “top-down” analysis typically reaches only ~35%.25,42 The incomplete fragmentation likely results from highly structured and disulfide bond-protected areas.41 The “bottom-up” method uses enzymatic antibody digestion followed by LC and tandem mass spectrometry (MS/MS) analysis to provide accurate mass values and product ions that imply the sequences of individual peptides. However, protein digestion typically requires several timeconsuming steps during which antibody modification may occur.43,44 Trypsin digestion, for instance, generally includes antibody denaturation and reduction followed by alkylation of thiol groups to prevent reforming of disulfide bonds. Moreover, proteolysis usually takes place at 37 ÂşC overnight, and basic digestion conditions favor deamidation of asparagine.45 Peptide coverage is frequently incomplete because of weak ionization efficiencies for some peptides along with loss of a few peptides during LC.46 Nevertheless, analysis of several digests catalyzed by different proteases often yields 100% sequence coverage.47 Recently, Srzentić et al. reported that digestion using the enzyme Sap 9 yields relatively large peptides (compared to tryptic digestion) and enables extended “bottom-up” LC-MS/MS analysis with nearly 100% peptide coverage for both light chain and heavy chains.26 Importantly, this enzyme functions in acidic conditions that limit deamidation and avoid the need for alkylation of cysteine. However, Sap9 must be recombinantly expressed and purified. 63 Analysis of larger peptides (3-20 kDa) obtained from limited digestion is usually termed a “middle-down” approach.48 Ultra-high resolution mass spectrometers can resolve the isotopic distributions of these peptides, and their relatively large size enhances the total peptide coverage (relative to tryptic digestion), which increases the sequence coverage when fragmentation is complete. Additionally, compared to “bottom-up” methods, the large size of “middle-down” peptides increases the probability that two or more PTMs will occur on the same peptide to enable correlation of these PTMs.49 In one “middle-down” strategy that yields peptides with masses around 25 kDa, papain cleaves antibodies at the hinge region and forms subunits such as Fab or F(ab’)2, but the digestion is sometimes difficult to control.11 Fornelli and coworkers employed the Immunoglobulin-degrading enzyme of Streptococcus (IdeS) to fragment Adalimumab into F(ab’)2 and Fc portions at the G-G bond below the hinge region.38 After reduction and denaturation, they obtained Fd, Lc and Fc/2 fragments, and analyzed these large peptides with LC-ETD MS/MS. Sequence coverage reached 70%. The large size of these peptides may still makes detection of deamidation difficult, and incomplete fragmentation limits sequence coverage. In addition to varying the digestion enzyme, limiting the digestion time may yield the large peptides required for “middle-down” protein characterization. Recently, Tan and coworkers adsorbed pepsin in the pores of nylon membranes and found that the lengths of proteolytic peptides from myoglobin and bovine serum albumin vary with the residence time of the protein solution in the membrane, i.e. shorter residence times generate longer peptides.50 This method can generate both “bottom-up” (long residence times) and “middle-down” (short residence times) peptides using a single enzyme, so control over digestion yields peptides with overlapping sequences. Small peptides give detailed sequence information, whereas larger peptides lead to 64 higher coverage. The large peptides may also contain many basic residues that lead to higher charge states, which is beneficial for ETD fragmentation.51 This research employs antibody proteolysis in pepsin-containing porous membranes to decrease the time and cost of digestion, increase both the peptide and sequence coverages in MS analysis, and limit antibody modification during digestion. Figure 2.1 shows the work flow for digestion and analysis. Porous nylon membranes and pepsin are inexpensive and readily available, and the high enzyme concentration in membrane pores allows digestion in a few minutes. Figure 2.1. Workflow for controlled digestion and analysis of antibodies. Acronyms: TCEPtris(2-carboxyethyl) phosphine, VH-variable region of the heavy chain; CH1, CH2, and CH3different constant regions of the heavy chain; CL-constant region of the light chain; VL-variable region of the light chain; Lc-light chain; Hc- heavy chain. Moreover, acidic digestion conditions limit deamidation and do not require protection of the thiol groups of cysteine. Other recent substrates for pepsin immobilization include aldehyde-modified polymethacrylate monoliths52 and fused-silica capillaries,53 but such supports do not readily afford the ms residence times available with in-membrane digestion. This study investigates inmembrane digestion of the entire antibody without separation of the light and heavy chains as 65 well as digestion of separated chains. Remarkably, digestion of 35 pmol of a reduced WatersTM antibody (WIgG1) occurs in less than 1 min with 100% peptide coverage of the light and heavy chains. We further demonstrate the benefits of this digestion strategy by comparing MS analyses of a monoclonal antibody light chain after digestion in a pepsin-containing membrane, after traditional in-solution digestion and using a “top-down” method. 2.2 Experimental 2.2.1 Materials A monoclonal immunoglobulin G was purchased from Waters (WIgG1, Intact mAb Mass Check Standard, 186006552), and Trastuzumab (Herceptin, Genentech) was dissolved in a phosphate buffer (KH2PO4 144 mg/L, NaCl 9000 mg/L, and Na2HPO4¡7H2O 795 mg/L, pH 7.4, Thermo Fisher) at a concentration of 21 mg/mL. Nylon membranes (LoProdyne LP, pore size 1.2 Îźm, 110 Îźm thickness) were acquired from Pall Corporation. The holder for membrane digestion (flangeless fitting system, Upchurch Scientific, A-424) was connected to 1/16 inch OD tubing via ferrules.50 Pepsin from porcine gastric mucosa (lyophilized powder, 3200-4500 units/mg protein), iodoacetamide (IAM, ≥99%), polystyrene sulfonate (PSS, average molecular weight ~70,000), and acetonitrile (ACN, HPLC grade, ≥99.9%) were obtained from Sigma Aldrich. Isopropyl alcohol (IPA, MACRON), sequencing grade modified trypsin (Promega), and trifluoroacetic acid (TFA, purchased from EMD) were used as received. Important chemicals for reduction and digestion include tris(2-carboxyethyl) phosphine hydrochloride (TCEP-HCl, >98%, Fluka), acetic acid (HOAc, Mallinckrodt, ACS), formic acid (>96%, Spectrum), and ammonium bicarbonate (Columbus Chemical). 66 2.2.2 Modification of Membranes with Pepsin We previously described pepsin-containing nylon membranes,50 but this work uses membranes with a nominal pore size of 1.2 Îźm instead of 0.45Âľm. The modification procedure includes sequential adsorption of PSS and pepsin, and the amount of pepsin adsorbed to the nylon membrane was estimated by determining the pepsin concentration in the loading solution before and after circulation through the membrane. A Nanodrop UV-Vis spectrometer (NanoDrop 2000, Thermo) measured the pepsin UV absorbance at 280 nm. 2.2.3 mAb Reduction and Characterization Antibodies were dissolved (WIgG1) or diluted (Trastuzumab) in deionized water to prepare 1 mg/mL stock solutions. The solution was stored at 4 ÂşC until use. For antibody reduction, 1 ÎźL of 0.1 M HOAc and 1 ÎźL of 0.1 M TCEP-HCl were added to 10 ÎźL of antibody stock solution, and this reaction mixture was incubated at 75 ÂşC for 15 min and finally diluted with 88 ÎźL of 5% FA. Alkylation of antibodies after reduction was conducted only prior to in-solution, tryptic digestion. In that case, 20 Îźg of WIgG1 was dried and reconstituted in 7 ÎźL of 2 mM TCEP-HCl solution prepared in 0.1 % HOAc containing 8 M urea. This mixture was incubated at 50 ÂşC for 10 min, and 7 ÎźL of 20 mM IAM in a 2 M NH4HCO3 solution containing 8 M urea was added. After incubation in the dark for 30 min, 6 ÎźL of 30 mM dithiothreitol in 100 mM NH4HCO3 solution containing 8 M urea was added. The reaction was incubated in the dark for 20 min to quench the IAM. Ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) confirmed separation of the light and heavy chains. Reduced antibody light and heavy chains were separated with an ACQUITY UPLC Protein BEH C4 column (1.7 Îźm diameter, 300Å pore size, 1 mm I.D. × 100 mm). UPLC 67 (Waters Open Architecture UPLC system) was performed at 40 ÂşC using a flow rate of 0.2 mL/min. In the gradient elution, solution A contained 0.1% FA in H2O, whereas solution B was 100% ACN. The applied gradient was 5 to 25% solution B in 2 min, 25 to 55% solution B in 12 min, and 55 to 90% solution B in 25 min. UPLC-ESI-QTOF-MS analysis of reduced antibody was conducted on a Waters Xevo G2-S QTOF mass spectrometer in positive-ion, sensitivity mode. The mass spectrometry parameters included: capillary voltage = 2.50 kV, sample cone voltage = 30.0 V, source offset = 12 V, source temperature = 100 ÂşC, desolvation temperature = 350 ÂşC, cone gas flow = 10 L/h, and desolvation gas flow = 600 L/h. The light-chain and heavychain signals were deconvoluted with the MaxEnt1 function in the MassLynx software. Parameters were set as follows: m/z input range = 400‒3000; output resolution = 0.2 Da/channel; output mass range = 22000-27000 Da for the light chain and 48000‒52000 Da for the heavy chain; uniform Gaussian width at half height of 0.5 Da for both the light and heavy chain; minimum intensity ratios of 50% for left and right; and a maximum of 10 iterations. Offline HPLC isolation of light and heavy chains (prior to digestion of these chains) was performed with a Shimadzu LC-20AB instrument equipped with a SPD-20AV ultraviolet detector. After reduction, the light and heavy chain mixture was injected onto a reversed phase TSKgel Protein C4-300 column (3.0 Îźm diameter, 300 Å pore size, 4.6 mm I.D. × 150 mm). LC was performed at 40 ÂşC using a flow rate of 1 mL/min. In the gradient elutions, solution A contained 0.05% TFA, 10% ACN, and 89.95% H2O, whereas solution B consisted of 0.045% TFA, 70% ACN, 9.955% IPA, and 20% H2O. Two different gradients were used to separate nonalkylated and alkylated chains. For nonalkylated antibody, the applied gradient was 0% to 30% solution B in 5 min, 30% to 60% solution B in 30 min, and 60% to 100% solution B in 5 min. For alkylated antibody, the gradient was 0% to 30% solution B in 5 min, 30% to 65% solution B in 35 min, and 65% to 100% 68 solution B in 10 min. UV absorbances at 215 nm and 280 nm were used to monitor the elution of antibody chains. Effluent was collected offline, dried with a SpeedVac, and analyzed by SDSPAGE with Coomassie Blue staining. 2.2.4 In-Membrane Digestion of Intact Antibody After reduction by TCEP, the nonalkylated mixture was passed through a pepsin-containing membrane in an Upchurch holder at flow rates of 0.13 or 130 mL/h using a syringe pump. The residence time was estimated assuming a nylon membrane porosity of 50% and an exposed membrane area of 0.02 cm2 (see equation (2-1) below). Hence, residence times were 3 s and 3 ms for 0.13 and 130 mL/h flow rates, respectively, and 100 ÎźL of effluent was collected for direct infusion MS analysis. 2.2.5 Digestion of the mAb Light and Heavy Chains 2.2.5.1 In-membrane digestion Ten Îźg of nonalkylated light chain (with the assumption that all of the light chain was recovered from the separation) was dissolved in 100 ÂľL of 2 mM TCEP in 5% FA solution. The mixture was heated at 80 ÂşC for 10 min, allowed to cool, and the light chain solution was passed through the membrane at flow rates of 0.13, 13 and 130 mL/h. Effluent was collected, dried with a SpeedVac and saved for MS analysis. The heavy chain was digested similarly. 2.2.5.2 In-solution digestion Four Îźg of alkylated light chain (with assumption that all of the light chain was recovered from the separation) was dissolved in 10 ÎźL of 2 mM TCEP solution containing 10 mM NH4HCO3. 69 The mixture was heated at 80 °C for 10 min, and cooled to room temperature. Two ÂľL of 0.1 Âľg/ÂľL trypsin solution was added to the mixture prior to incubation at 37 ÂşC for 16 h. The reaction was quenched by addition of 5 ÎźL of acetic acid, immediately frozen with liquid nitrogen, and dried with a SpeedVac before reconstitution for MS. 2.2.6“Top-down” Analysis of a mAb Light Chain An Agilent Technologies (Palo Alto, CA) 1100 Series binary HPLC system was interfaced with a Thermo Fisher Scientific Orbitrap EliteTM Hybrid Ion Trap-Orbitrap Mass Spectrometer (San Jose, CA) for online separation of TCEP-reduced WIgG1. About 100 fmol of reduced WIgG1 was pressure-loaded onto a fused silica capillary column (75 Âľm I.D. × 360 Âľm O.D.) packed with 10 cm of Agilent POROSHELL 300SB-C18 particles (5 Îźm diameter, 300 Å pore size). The back end of the column was equipped with a laser-pulled nanoelectrospray emitter tip,54 and the column was initially rinsed for 10 min with 0.3% formic acid in water to remove salts. Protein sample was eluted at 60 ÂşC at a flow rate of 100 nL/min using the following gradient: 030% B for 5 min, 30-50% B for 20 min, 50-100% B for 5 min. Solution A contained 0.3% FA in H2O, whereas solution B consisted of 0.3% FA, 72% ACN, 18% IPA and 9.7% H2O. 2.2.7 Mass Spectrometry and Data Analysis After drying with a Speedvac, in-membrane and in-solution digests were reconstituted in 1% acetic acid, 49% H2O, and 50% methanol, loaded into a Whatman multichem 96-well plate (Sigma Aldrich) and sealed with Teflon Ultrathin Sealing Tape (Analytical Sales and Services, Prompton Plains, NJ). An Advion Triversa Nanomate nanoelectrospray ionization (nESI) source (Advion, Ithaca, NY) was used to introduce the sample into a high-resolution accurate mass Thermo Fisher Scientific LTQ Orbitrap VelosTM mass spectrometer (San Jose, CA) equipped 70 with a dual pressure ion trap, HCD cell, and ETD. The spray voltage and gas pressure were set to 1.4 kV and 1.0 psi, respectively. The ion source interface had an inlet temperature of 200 °C with an S-Lens value of 65%. High-resolution mass spectra were acquired in positive ionization mode across the m/z range of 300-2000 using the FT analyzer operating at 100 000 mass resolving power. Spectra were the average of 100 scans. Mass spectra were deconvoluted using the Xtract function of the XCalibur software. Proteolytic peptide identification and CID/HCD/ETD MS/MS data analysis were performed manually (for isotopic distributions with signal to noise >5) by matching MS and MS/MS product ions with data generated in silico using ProteinProspector (v 5.14.1 University of California, San Francisco). Mass tolerance was set for 5 ppm. “Top-down” MS analyses of the WIgG1 light chain include a full MS scan at m/z 300-2000 in the Orbitrap at 240,000 mass resolving power, and three MS/MS scans (5-ms ETD, 15-ms ETD, and CID targeted on the +28 charge-state Lc ion at m/z 865.2 with a 3 m/z isolation window) in the Orbitrap at 120,000 mass resolving power (5 microscans per MS/MS scan). The Lc-targeted ETD (13 scans) or CID (9 scans) MS/MS spectra were merged and extracted from the raw file using Xcalibur™ 2.1 (Thermo Scientific). Each extracted ETD or CID spectrum was then searched against the sequence of WIgG1 (provided by Waters) using ProSightPC 3.0. Search parameters included: 5 Da precursor tolerance (monoisotopic), 15 ppm fragment tolerance (monoisotopic), ∆m mode on, and disulfide off. The c-, z-, b- and y-type fragment ions assigned by ProSightPC 3.0 were manually verified before acceptance. 71 2.3 Results and discussion 2.3.1 Protease-containing Membranes Figure 2.1 shows the workflow we employ to analyze mAbs using in-membrane digestion. The procedure exploits enzyme-containing membranes that we prepare using sequential adsorption of PSS and pepsin in nylon membranes at pH 2.3. PSS adsorption provides a negatively charged surface that captures pepsin, which is positively charged at low pH. Similar to other membrane modifications through electrostatic adsorption, protease immobilization should occur throughout the membrane.55 These reactors are very active because of the high local enzyme concentration in membrane pores.50 The extent of digestion varies with the solution residence time in the membrane, tres, which is a function of the membrane thickness, l, the volumetric flow rate, Q, the exposed area at the faces of the membrane, A, and the membrane porosity, Îľ (equation (2-1)). tres=lAÎľ/Q (2-1) Our prior study used a membrane with nominal 0.45 Âľm pores and a thickness of 170 Âľm,50 whereas this work employs both a larger pore size (1.2 Âľm) and a lower thickness (110 Âľm) to further limit digestion and provide longer peptides. The lower thickness decreases the residence time for a given flow rate, and the larger pore size should give longer radial diffusion distances to immobilized enzymes. Analysis of the pepsin loading solution before and after circulating through the membrane suggests an immobilized pepsin concentration of ~70 mg per mL of membrane. 72 2.3.2 mAb Reduction and Characterization Effective antibody digestion requires reduction of disulfide bonds to give the protease access to cleavage sites. We employ TCEP as a reducing agent because it can function under acidic conditions that both prevent reformation of disulfide bonds and partially denature the antibody. Thus, acidic conditions avoid the need for urea denaturation and alkylation. Because pepsin is enzymatically active at pH 2-3, peptic digests are compatible with ESI-MS without further purification. UPLC-ESI-QTOF-MS analysis verified the separation of light and heavy chains after TCEP reduction (Figure 2.2). Based on deconvolution using MaxEnt1 software, the average mass of the antibody light chain is 24198.0, which agrees with the theoretical mass of 24197.7 provided by the manufacturer. The deconvoluted heavy chain mass spectrum shows three glycoforms with mass differences of 162 Da. 73 Figure 2.2. mAb Reduction and Characterization. (A) Chromatogram showing the UPLC separation of the WIgG1 light and heavy chains. (B) Mass spectrum at a retention time of 5.85 min showing the light chain isotopic envelope. The inset is the deconvoluted mass spectrum. (C) Mass spectrum at a retention time of 7.51 min showing the heavy chain isotopic envelope. The inset is the deconvoluted mass spectrum. See Figure 2.9 for glycoform assignments. 74 2.3.3 mAb Digestion in Membranes In-membrane digestion occurs during passage of a reduced-antibody solution through a pepsincontaining membrane. At a flow rate of 130 mL/h, which corresponds to a 3-ms residence time, digestion of a 100-ÂľL solution requires less than one minute. As Figure 2.3 shows, the infusion mass spectrum of digested WIgG1 contains signals from two large peptides, L1-140 (light-chain amino acids 1-140) and L141-219, which cover the entire light-chain sequence. L1-140 shows consecutive charge distribution isotopic envelopes from +8 to +14, and this large peptide (15 kDa) covers the CDR-L1, CDR-L2 and CDR-L3 regions of the antibody. Figure 2.3. Part of the mass spectrum of an in-membrane digest (3-ms residence time) of reduced WIgG1 antibody. The labeled signals show the charge-state distributions of peptides containing the amino acids 1-140 (purple), and 141-219 (red) of the light chain. These two large peptides cover the entire light chain sequence. 75 Similarly, the other large peptide with light-chain amino acids 141-219 shows multiple charge states from +6 to +13. These charge states correspond well with the number of basic residues in the peptides. L1-140, for instance, includes 13 basic residues, 6 K, 5 R, and 2 H, which explains the highest charge state of +14, considering that the N-terminus also can capture one proton. L141-219 contains 12 basic residues, 7 K, 3 R, and 2 H, and the highest charge state is +13. Some of the other abundant signals in the mass spectrum of reduced antibody (see Figure 2.4) result from peptides whose sequences overlap with these two large peptides, such as amino acids L1-51, L1-75, L1-90, L76-140, L91-140, L141-165, and L166-219. Combinations of these peptides also cover the entire light chain sequence. The mass spectrum of the reduced-antibody digest (Figure 2.4) also shows many peptides from the heavy chain, such as H1-104, H105-113, H114-179, H180-235, H236-273, H274-363, H364399, and H400-441. H1-104 covers CDR-H1 and CDR-H2 regions, and shows N-terminal pyroglutamic acid formation (-17 Da compared to the mass of the original sequence). The heavychain peptide coverage (percentage of amino acids comprised by the detected peptides) for the 3ms digestion is 100%. 76 Figure 2.4. Part of the mass spectrum of a 3-ms, in-membrane digest of WIgG1. Labels show the amino acids on the Hc and Lc. Black circles denote unidentified impurities with +1 charge states. 77 Figure 2.4 (cont’d) 78 Figure 2.4 (cont’d) 79 Figure 2.4 (cont’d) 80 Replicate 3-ms, in-membrane digestions with 3 different membrane pieces show similar signal intensities (Figure 2.5). In contrast to ms digestion, 3-s in-membrane digestion (slower flow through the membrane) shows shorter peptides from the Lc and Hc (see Tables 2.1 and 2.2 for lists of peptides). Figure 2.5. Mass spectra of 3 different 3-ms, in-membrane digests of WIgG1. Each digestion employed a different piece of membrane cut from a larger membrane with a diameter of 25 mm. For 18 out of the 20 peptides that give the highest signal intensities, standard deviations of signals (relative to the most intense peak in the spectrum) from the three digests are <3% of the average signal. All of the relative standard deviations are <10%. 81 Table 2.1. Light- and heavy-chain peptides identified from a 3-ms in-membrane digest of WIgG1. m/z of [M+H]+ 5649.8334 8177.0736 8324.1442 9834.9742 9998.0410 15050.5984 2546.2594 1676.9197 6892.5108 5234.5978 2947.5393 9152.2863 6223.7777 2120.0518 11392.6549 19131.4846 2958.4110 1042.4996 6734.4204 4275.1546 5880.8620 1624.7200 Peptide Sequence (-)DVLMTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEWY LQKPGQSPKL(L) (-)DVLMTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEWY LQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTD(F) (-)DVLMTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEWY LQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDF(T) (-)DVLMTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEWY LQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA EDLGV(Y) (-)DVLMTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEWY LQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA EDLGVY(Y) (-)DVLMTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEWY LQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA EDLGVYYCFQGSHVPLTFGAGTKLEIKRADAAPTVSIFPPSSEQ LTSGGASVVCF(L) (L)LIYKVSNRFSGVPDRFSGSGSGTD(F) (D)FTLKISRVEAEDLGV(Y) (D)FTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLEIKRADA APTVSIFPPSSEQLTSGGASVVCF(L) (V)YYCFQGSHVPLTFGAGTKLEIKRADAAPTVSIFPPSSEQLTS GGASVVCF(L) (F)LNNFYPKDINVKWKIDGSERQNGVL(N) (F)LNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSM SSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(-) (L)NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTS TSPIVKSFNRNEC(-) (E)ATHKTSTSPIVKSFNRNEC(-) (-)QVQLKESGPGLVAPSQSLSITCTVSGFSLLGYGVNWVRQP PGQGLEWLMGIWGDGSTDYNSALKSRISITKDNSKSQVFLKM NSLQTDDTAKYYCTRAPYGKQY(F) (-)QVQLKESGPGLVAPSQSLSITCTVSGFSLLGYGVNWVRQP PGQGLEWLMGIWGDGSTDYNSALKSRISITKDNSKSQVFLKM NSLQTDDTAKYYCTRAPYGKQYFAYWGQGTLVTVSAAKTTPP SVYPLAPGSAAQTDSMVTLGCLVKGYFPEPVTVTWNSGSLSSG VHTFPAVLQSDL(Y) (F)LKMNSLQTDDTAKYYCTRAPYGKQY(F) (Y)FAYWGQGTL(V) (L)VTVSAAKTTPPSVYPLAPGSAAQTDSMVTLGCLVKGYFPEP VTVTWNSGSLSSGVHTFPAVLQSDL(Y) (L)YTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRD(C) (L)YTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCG CKPCICTVPEVSSV(F) (D)CGCKPCICTVPEVSSV(F) 82 Amino Acids L1-51 L1-75 L1-76 L1-90 L1-91 L1-140 L52-75 L76-90 L76-140 L91-140 L141-165 L141-219 L166-219 L201-219 H1-104 H1-179 H80-104 H105-113 H114-179 H180-219 H180-235 H220-235 Table 2.1 (cont’d) 1401.8122 2358.3482 4349.3218 2009.9866 11662.6778* 11824.7464** 6979.6366 4249.8985 4720.2686 3982.8622 2507.1853 (V)FIFPPKPKDVLT(I) (V)FIFPPKPKDVLTITLTPKVTC(V) (V)FIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWF(V) (C)VVVDISKDDPEVQFSWF(V) (F)VDDVEVHTAHTQPREEQFNSTFRSVSELPIMHQDWLNGKEF KCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKV SLTCM(I) (F)VDDVEVHTAHTQPREEQFNSTFRSVSELPIMHQDWLNGKEF KCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKV SLTCM(I) (L)PIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQ VYTIPPPKEQMAKDKVSLTCM(I) (M)ITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSY(F) (Y)FVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHS PG(-) (L)NVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG(-) (F)TCSVLHEGLHNHHTEKSLSHSPG(-) H236-247 H236-256 H236-273 H257-273 H274-363 H274-363 H302-363 H364-399 H400-441 H406-441 H419-441 * 11662.6778 is the monoisotopic mass for H274-363 with G0F glycosylation. ** 11824.7464 is the monoisotopic mass for H274-363 with G1F glycosylation. Table 2.2. Light- and heavy-chain peptides identified from a 3-s in-membrane digest of WIgG1. m/z of [M+H]+ Peptide Sequence 1217.6434 (-)DVLMTQTPLSL(P) (-)DVLMTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEW YLQKPGQSPKL(L) (-)DVLMTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEW YLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTD(F) (-)DVLMTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEW YLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDF(T) (-)DVLMTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEW YLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE AEDLGV(Y) (-)DVLMTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEW YLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE AEDLGVY(Y) (L)MTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEWYLQK PGQSPKL(L) (L)MTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEWYLQK PGQSPKLLIYKVSNRFSGVPDRFSGSGSGTD(F) (L)MTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEWYLQK PGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDF(T) (L)PVSLGDQASISCRSSQ(Y) (L)PVSLGDQASISCRSSQYIVHSNGNTYLEWYLQKPGQSPKL( L) 5649.8334 8177.0736 8324.1442 9834.9742 9998.0410 5322.6528 7849.8865 7996.9674 1634.7802 4451.2054 83 Amino Acids L1-11 L1-51 L1-75 L1-76 L1-90 L1-91 L4-51 L4-75 L4-76 L12-27 L12-51 Table 2.2 (cont’d) 6978.4512 2835.4426 5362.6873 1258.7134 3785.9593 2546.2606 2693.3298 4204.1648 1676.9197 1839.9848 1529.8510 1692.9145 2089.0210 5234.5978 1925.9566 5071.5334 3164.5924 2947.5393 7051.2610 9152.2863 4122.7438 6223.7705 1988.8645 4089.8950 2120.0518 3405.6730 6345.0612 3217.6082 2223.2054 1838.0098 2210.2288 4777.4020 1353.6991 1725.9178 4293.0918 1627.7971 2958.4106 (L)PVSLGDQASISCRSSQYIVHSNGNTYLEWYLQKPGQSPKLL IYKVSNRFSGVPDRFSGSGSGTD(F) (Q)YIVHSNGNTYLEWYLQKPGQSPKL(L) (Q)YIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFS GSGSGTD(F) (W)YLQKPGQSPKL(L) (W)YLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTD(F) (L)LIYKVSNRFSGVPDRFSGSGSGTD(F) (L)LIYKVSNRFSGVPDRFSGSGSGTDF(T) (L)LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGV(Y) (D)FTLKISRVEAEDLGV(Y) (D)FTLKISRVEAEDLGVY(Y) (F)TLKISRVEAEDLGV(Y) (F)TLKISRVEAEDLGVY(Y) (V)YYCFQGSHVPLTFGAGTKL(E) (V)YYCFQGSHVPLTFGAGTKLEIKRADAAPTVSIFPPSSEQLTS GGASVVCF(L) (Y)YCFQGSHVPLTFGAGTKL(E) (Y)YCFQGSHVPLTFGAGTKLEIKRADAAPTVSIFPPSSEQLTSG GASVVCF(L) (L)EIKRADAAPTVSIFPPSSEQLTSGGASVVCF(L) (F)LNNFYPKDINVKWKIDGSERQNGVL(N) (F)LNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYS MSSTLTLTKDEYERHNSYTCE(A) (F)LNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYS MSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(-) (L)NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE(A) (L)NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC(-) (L)TLTKDEYERHNSYTCE(A) (L)TLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(-) (E)ATHKTSTSPIVKSFNRNEC(-) (L)MGIWGDGSTDYNSALKSRISITKDNSKSQVF(L) (L)MGIWGDGSTDYNSALKSRISITKDNSKSQVFLKMNSLQTD DTAKYYCTRAPYGKQY(F) (G)IWGDGSTDYNSALKSRISITKDNSKSQVF(L) (Y)NSALKSRISITKDNSKSQVF(L) (L)KSRISITKDNSKSQVF(L) (L)KSRISITKDNSKSQVFLKM(N) (L)KSRISITKDNSKSQVFLKMNSLQTDDTAKYYCTRAPYGKQ Y(F) (I)SITKDNSKSQVF(L) (I)SITKDNSKSQVFLKM(N) (I)SITKDNSKSQVFLKMNSLQTDDTAKYYCTRAPYGKQY(F) (F)LKMNSLQTDDTAKY(Y) (F)LKMNSLQTDDTAKYYCTRAPYGKQY(F) 84 L12-75 L28-51 L28-75 L41-51 L41-75 L52-75 L52-76 L52-90 L76-90 L76-91 L77-90 L77-91 L91-109 L91-140 L92-109 L92-140 L110-140 L141-165 L141-200 L141-219 L166-200 L166-219 L185-200 L185-219 L201-219 H49-79 H49-104 H51-79 H60-79 H64-79 H64-82 H64-104 H68-79 H68-82 H68-104 H80-93 H80-104 Table 2.2 (cont’d) 2586.1906 1349.6284 1042.4996 2646.3451 3233.6629 6290.2188 3662.8918 3076.5775 3519.7810 4275.1566 5880.8620 1624.7196 1300.7644 1401.8122 2358.3478 1141.6604 1076.6008 975.5532 989.5138 1442.7366 2009.986 4702.0793* 4864.1330** 5026.1942*** 1467.7027 4718.5089 6979.6366 5530.9434 2280.1402 1425.6776 2843.2366 756.4278 4720.2686 3982.8598 2507.1853 2406.1318 2303.1238 2216.0888 2003.9402 (M)NSLQTDDTAKYYCTRAPYGKQY(F) (Y)YCTRAPYGKQY(F) (Y)FAYWGQGTL(V) (L)VTVSAAKTTPPSVYPLAPGSAAQTNSM(V) (L)VTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCL(V) (L)VTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPE PVTVTWNSGSLSSGVHTFPAVL(Q) (M)VTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVL(Q) (L)VKGYFPEPVTVTWNSGSLSSGVHTFPAVL(Q) (L)VKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDL(Y) (L)YTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRD(C ) (L)YTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC GCKPCICTVPEVSSV(F) (D)CGCKPCICTVPEVSSV(F) (V)FIFPPKPKDVL(T) (V)FIFPPKPKDVLT(I) (V)FIFPPKPKDVLTITLTPKVTC(V) (I)FPPKPKDVLT(I) (L)TITLTPKVTC(V) (T)ITLTPKVTC(V) (C)VVVDISKDD(P) (C)VVVDISKDDPEVQ(F) (C)VVVDISKDDPEVQFSWF(V) (F)VDDVEVHTAHTQPREEQFNSTFRSVSEL(P) (F)VDDVEVHTAHTQPREEQFNSTFRSVSEL(P) (F)VDDVEVHTAHTQPREEQFNSTFRSVSEL(P) (L)PIMHQDWLNGKE(F) (L)PIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQ V(Y) (L)PIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQ VYTIPPPKEQMAKDKVSLTCM(I) (E)FKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAK DKVSLTCM(I) (V)YTIPPPKEQMAKDKVSLTCM(I) (M)ITDFFPEDITVE(W) (E)WQWNGQPAENYKNTQPIMDTDGSY(F) (Y)FVYSKL(N) (Y)FVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHS PG(-) (L)NVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG(-) (F)TCSVLHEGLHNHHTEKSLSHSPG(-) (T)CSVLHEGLHNHHTEKSLSHSPG(-) (C)SVLHEGLHNHHTEKSLSHSPG(-) (S)VLHEGLHNHHTEKSLSHSPG(-) (L)HEGLHNHHTEKSLSHSPG(-) 85 H83-104 H94-104 H105-113 H114-140 H114-146 H114-175 H141-175 H147-175 H147-179 H180-219 H180-235 H220-235 H236-246 H236-247 H236-256 H238-247 H247-256 H248-256 H257-265 H257-269 H257-273 H274-301 H274-301 H274-301 H302-313 H302-343 H302-363 H314-363 H344-363 H364-375 H376-399 H400-405 H400-441 H406-441 H419-441 H420-441 H421-441 H422-441 H424-441 * 4702.0793 show monoisotopic mass of H274-301 with G0F glycosylation. ** 4864.1330 show monoisotopic mass of H274-301 with G1F glycosylation. *** 5026.1942 show monoisotopic mass of H274-301 with G2F glycosylation. As a second example, we digested Trastuzumab (Herceptin), a commercial humanized mAb for breast cancer treatment. Detailed characterization of Trastuzumab is crucial for quality control, and several groups recently presented MS-based interrogation of this antibody.3,37,56-60 Remarkably, a 3-ms digestion of reduced Trastuzumab followed by MS analysis yields 100% peptide coverage for both the light and heavy chains. (See Figure 2.6 for the mass spectrum.) A large peptide, L1-83, covers the CDR-L1 and CDR-L2 regions. Moreover, a peptide containing amino acids 1-115 on the heavy chain contains all the CDR-H regions. These data demonstrate that rapid digestion (less than 1-min total digestion time) with a pepsin-containing membrane can yield essentially complete peptide coverage. Moreover, the simple acidic reduction procedure requires only 15 min, and the MS data collection takes 5 min when using direct infusion. 86 Figure 2.6. Part of the mass spectrum of a 3-ms, in-membrane digest of Trastuzumab. Labels show the amino acids on the Hc and Lc. Black circles denote unidentified impurities with +1 charge states. 87 Figure 2.6 (cont’d) 88 Figure 2.6 (cont’d) 89 Figure 2.6 (cont’d) 90 2.3.4 mAb Light-Chain Analysis using In-membrane or Tryptic In-solution Digestion To further investigate antibody sequence information and compare “middle-down” and “bottomup” methods, we analyzed the WIgG1 light chain after its isolation using HPLC. SDS-PAGE showed the separation of light and heavy chains, and HPLC retention times guided offline collection of the desired fractions. We reconstituted the antibody light chain in a lowconcentration TCEP solution to prevent reformation of disulfide bonds. Figure 2.7. Deconvoluted ESI-Orbitrap mass spectra of the WIgG1 light chain digested with 3-ms (A) and 3-s (B) residence times in a pepsin-containing membrane. Other small peptides appear at lower m/z values. Deconvoluted peaks (generated by Xtract) represent m/z values of peptides with a +1 charge. 91 Figure 2.7 shows the deconvoluted mass spectra of the WIgG1 light chain digested with 3-ms and 3-s residence times in the pepsin-containing membrane. Upon increasing the digestion residence time, large peptides undergo additional cleavages to give smaller peptides. Dominant signals from the 3-ms digestion stem from large peptides, such as L52-90, L91-140, L1-51, L166-219, L76-140, L4-75, L1-75 and L141-219. In contrast, 3-s digestion yields smaller peptides such as L41-51, L52-75, and L76-90, and in several cases combinations of the small peptides give the larger peptides from the 3-ms digestion. The 3-ms digestion enables 100% peptide coverage of the light chain, whereas the 3-s digestion shows only 95% coverage due to the absence of amino acids 1-11. The lack of basic amino acids in the first 11 residues (see Figure 2.9 below for the light-chain sequence) may explain the absence of peptide coverage in this region. We also performed an in-solution, tryptic digestion of the alkylated light chain for comparison. Eighteen tryptic peptides cover the entire Lc sequence. These peptides show an average length of 12 amino acids, which agrees with the theoretical tryptic peptide length, 8-25 residues. Table 2.3 gives details of the tryptic peptide m/z values. 2.3.5 Comparison of Light-chain Sequence Coverage Using “Middle-down”, “Bottom-up” and “Top-down” methods Specific amino acid sequence information requires MS/MS data with extensive fragmentation. While larger peptides may contain multiple CDRs and give high peptide coverage, small peptides are easier to fragment in some MS/MS methods and may provide higher sequence coverage. CID is widely used in “bottom up” LC-MS/MS proteomics and gives the peptide sequence information via a series of b- and y- ions. One of the peptides, L52-75, from a 30-ms, 92 in-membrane digestion of the WIgG1 light chain covers the entire CDR-L2 region. CID-MS/MS of this peptide shows 100% sequence coverage, i.e. cleavage of all amide bonds (Figure 2.8). Table 2.3. WIgG1 peptides identified from an in-solution tryptic digest of the alkylated light chain. m/z of [M+H]+ 2588.2982 3024.4741 649.4264 475.2607 777.3866 1303.6118 375.2338 2845.3390 502.3219 658.4225 3727.8322 588.3333 990.4974 1591.7294 1534.7250 711.2921 1347.5758 832.4749 926.3756 Peptide Sequence (-)DVLMTQTPLSLPVSLGDQASISC(Carbamidomethyl)R(S) (R)SSQYIVHSNGNTYLEWYLQKPGQSPK(L) (K)LLIYK(V) (K)VSNR(F) (R)FSGVPDR(F) (R)FSGSGSGTDFTLK(I) (K)ISR(V) (R)VEAEDLGVYYC(Carbamidomethyl)FQGSHVPLTFGAG TK(L) (K)LEIK(R) (K)LEIKR(A) (K)RADAAPTVSIFPPSSEQLTSGGASVVC(Carbamidomethy l)FLNNFYPK(D) (K)DINVK(W) (K)WKIDGSER(Q) (R)QNGVLNSWTDQDSK(D) (K)DSTYSMSSTLTLTK(D) (K)DEYER(H) (R)HNSYTC(Carbamidomethyl)EATHK(T) (K)TSTSPIVK(S) (K)SFNRNEC(Carbamidomethyl)(-) Amino Acids L1-24 L25-50 L51-55 L56-59 L60-66 L67-79 L80-82 L83-108 L109-112 L109-113 L113-147 L148-152 L153-160 L161-174 L175-188 L189-193 L194-204 L205-212 L213-219 However, not all peptides show such complete fragmentation. One limitation of quadrupole iontrap CID is the loss of fragment ions in the low m/z range due to the low-mass cutoff determined by the radio frequency amplitude. In contrast, HCD in the LTQ Orbitrap VelosTM mass spectrometer does not have this limitation and facilitates identification of N and C terminal fragment ions.61 MS/MS analyses of 12 proteolytic peptides L1-51, L4-51, L12-51, L24-51, L5275, L76-90, L91-136, L110-136, L91-140, L141-165, L166-200, and L201-219 were conducted using both CID and HCD. Combining CID and HCD gives higher sequence coverage than CID 93 alone. For instance, CID of L141-165 does not break the Pro-Lys bond, while HCD cleaves this bond and gives 100% sequence coverage of this peptide Figure 2.8. CID-MS/MS spectrum of the WIgG1 light-chain peptide L52-75, which covers the entire CDR-L2 region. The sequence at the top of the figure denotes the formation of b and y ions (only some of the b and y ions are labeled in the spectrum). CID and HCD of L52-75 and L76-90 give 100% sequence coverage of L52-90. Relatively large peptides produced by 3-ms light-chain digestion, L1-51 and L91-140, were fragmented by ETD. In contrast to CID, which cleaves the labile bonds on the peptide chain, ETD induces fragmentation in a sequence-independent manner. A low residue/charge ratio results in effective 94 ETD fragmentation, so we chose the highest charge states of each peptide for ETD analysis, (+5 for L1-51 and for L91-140). The c- and z- ions produced by ETD (of L1-51 and L91-140) along with the b- and y- ions from CID and HCD of the 12 peptides mentioned previously give 99% bond cleavages in the light chain. Only three amide bond linkages did not dissociate, 25S-26S, 100P-101L, and 113R-114A. Figure 2.9 gives a summary of the cleavage sites. Figure 2.9 Summary of bond cleavage sites from CID, HCD and ETD-MS/MS of WIgG1 light-chain peptides obtained from a 30-ms digestion in pepsin-containing membranes. Red and purple letters cover the light-chain variable region (VL), with purple letters denoting complementarity determining regions (CDRs). Green letters represent the light-chain constant region (CL). The figure does not show redundant cleavages sites from c and z ions. For comparison, we fragmented the 18 alkylated light-chain tryptic peptides (mentioned in the last section), using CID. The MS/MS spectra reveal cleavage of 205 out of 218 bonds via either enzyme cleavage or CID-MS/MS, which gives 94% sequence coverage. We also conducted HCD on these peptides to obtain nine more fragmentation sites. The combination of CID, HCD, and tryptic cleavage sites yields 99% sequence coverage (Figure 2.10). Notably, with in-solution 95 tryptic digestion, the CDR-L2 region spans three peptides, 51-55, 56-59, and 60-66 making direct characterization of this region difficult. Figure 2.10. Summary of the bond cleavage sites from CID and HCD-MS/MS of peptides obtained from tryptic, in-solution diges-tion of the WIgG1 Lc. We also performed ETD and CID for the entire reduced WIgG1 light chain. Figure 2.11 shows Orbitrap MS/MS spectra from online LC-MS analysis with 5-ms ETD, 15-ms ETD, and CID with default energy, respectively. Figure 2.12 gives an example of “top-down” ETD MS/MS showing partial sequence coverage. Direct ETD (combining the results from 5-ms and 15-ms ETD) of the light chain generated 53 and 50 detectable c- and z-type ions, respectively; and CID yielded 9 and 25 detectable b- and y-type ions, respectively. These c-, z-, b-, and y-type fragment ions collectively give 120 unique gas-phase backbone cleavages (Figure 2.13), which corresponds to 55% sequence coverage of the WIgG1 light chain. 96 Figure 2.11 “Top-Down” HPLC MS/MS spectra of the WIgG1 light chain. The MS/MS parameters were 15-ms ETD (A, 13 scans merged), 5-ms ETD (B, 13 scans merged), and CID (C, 9 scans merged), respectively. The precursor ion isolation window was 865.2Âą1.5 m/z. The FTMS MSn automatic gain control (AGC) target was set at 3 x 105 (A and B) or 1 x 105 (C), and the ETD reagent AGC target was set at 3 x 105. Each MS/MS scan included 5 microscans. 97 Figure 2.12. Orbitrap FT MS/MS spectrum after Xtract deconvolution of the original MS/MS spectrum (Figure 2.11A) resulting from 15-ms ETD of the WIgG1 Lc. Fragment c- and z- ions are labeled with black and red colors, respectively. The Nterminal (black) and C-terminal (red) sequences of the WIgG1 light chain are placed above the corresponding c- and z- ions, with the fragmentation sites indicated by dashed lines. The dashed lines labeled with * stem from c- or z-ions found from the original MS/MS spectrum, these ions are not displayed in this figure due to incomplete Xtract deconvolution. 98 Figure 2.13. Cleavage sites in “top-down” analysis of the antibody light chain. The sequence coverage is 55% after combining CID, 5-ms ETD and 15-ms ETD. 2.3.6 Detecting PTMs on the light and heavy chains PTMs affect antibody activities in a variety of ways and are commonly introduced to the sequence during manufacture, purification and storage. Hu et al. showed that oxidation on the light chain may induce a structural change and destabilize the protein.62 We compared two batches of WIgG1 by conducting 30-ms digestions of their light chains and performing MS analysis. Figure 2.14 compares the signals for peptide L166-219 (+5 charge state) for the two batches of antibody. For the second batch of antibody, the spectrum shows two strong isotopic envelopes whose deconvoluted mass difference is 15.9980, suggesting a Met oxidation. Because the spectra for the two batches of antibody were obtained under the same analysis conditions, this result suggests that oxidation does not occur significantly during ESI. Further MS/MS analysis of these two peptides from the second batch of antibody confirms that oxidation occurs at M 180. (See Figure 2.15 for MS/MS spectra). A similar strategy revealed M 393 oxidation (Figure 2.16) and N 138 deamidation on the heavy chain (Figure 2.17). 99 Figure 2.14. Part of the ESI-Orbitrap mass spectra of reduced WIgG1 light chains after digestion for 30-ms in pepsin-containing membranes. The top and bottom spectra come from two batches of antibody, and the largest isotopic envelopes represent the +5 charge state of the peptide containing amino acids L166-219. Figure 2.15. Part of the CID-MS/MS spectra of light-chain amino acids L166-219 (top) and oxidized L166-219 (bottom), demonstrating the oxidation at methionine 180. Peptides were obtained from 30-ms, in-membrane digestion of the light chain. 100 Figure 2.16. Part of the ESI-Orbitrap mass spectrum of a reduced WIgG1 heavy chain after in-membrane digestion with a residence time of 3 sec. The labeled isotope envelopes stem from the peptide H372-399 with (right) and without (left) oxidation. Another important PTM, glycosylation, plays an important role for binding to the Fc receptor and, thus, affects antibody-dependent cell-mediated cytotoxicity. A 3-s digestion of the heavy chain produced peptides with amino acids H274-301, and these peptides show a clear mass distribution characteristic of three glycoforms (Figure 2.18). This result matches with characterization of the entire heavy chain by LC-ESI-Q-TOF MS (Figure 2.2). Overall, these data show that rapid, in-membrane digestion enables facile characterization of mAb PTMs. 101 Figure 2.17. MS and CID-MS/MS spectra of the heavy-chain peptide H114-140 from a 3-s, in-membrane digestion. The spectra reveal deamidation on N138. (A) Part of the ESI-Orbitrap mass spectrum. The signals stem from the peptide H114-140 (red labels), and the same peptide with N138 deamidation (purple labels). (B) HCD-MS/MS reveals N138 deamidation. Insets show expanded regions of the spectrum. 102 Figure 2.18. Part of the ESI-Orbitrap mass spectrum of a reduced WIgG1 heavy chain after digestion with a 3-s residence time in a pepsin-containing membrane. The labeled isotopic envelopes result from the peptide H274-301 (+4 charge state), which contains 3 N292 glycoforms separated by 162 Da intervals due to galactose units. The inset shows the spectrum that results from deconvolution of the mass range in the spectrum. Unlabeled peaks stem from other peptides. Drawings represent the different glycoforms with Gal=galactose, GlcNac=Nacetylglucosamine, Man=mannose, and Fuc=fucose. 2.4 Conclusion This work used pepsin-modified membranes as controlled reactors for antibody proteolysis prior to MS analysis. Pepsin is an inexpensive protease that enables membrane digestion in acidic conditions, which avoids the need for antibody alkylation and minimizes oxidation during 103 digestion. Moreover, the high local enzyme concentration in membrane pores affords digestion of 100 ÎźL of antibody solution in less than one minute. Variation of the residence times of reduced antibody solutions in the membranes yields “bottom-up” (1-2 kDa) to “middle-down” sized peptides (5-15 kDa) for the light and heavy chains, and these peptides cover the entire antibody sequence. As needed, digestion with different flow rates can enhance sequence coverage. Analysis of the light-chain and heavy-chain proteolytic peptides reveals sites for oxidation, deamidation and N-terminal pyroglutamic acid formation as well as glycosylation patterns. Furthermore, 30-ms in-membranes proteolysis of the light-chain followed by CID, HCD and ETD MS/MS of peptic peptides cleaves 99% of the amino acid bonds in the light chain. Traditional in-solution tryptic digestion of the light chain combined with CID and HCD-MS/MS also gives 99% sequence coverage, whereas “top-down” analysis of the entire light chain by CID, 5-ms, and 15-ms ETD shows a sequence coverage of 55%. With minimal sample preparation time, membrane digestion leads to high peptide and sequence coverages for identification of PTMs by MS. 2.5 Acknowledgement We gratefully acknowledge the US National Science Foundation (CHE-1152762 and CHE1506315) for funding this work. We thank Dr. Mohammad Muhsin Chisti from Michigan State Uni-versity for providing Trastuzumab. We also thank the Michigan State University Mass Spectrometry Facility and Dr. Todd Lydic from the Molecular Metabolism and Disease Collaborative Mass Spectrometry Core for helping analyze the samples. 104 REFERENCES 105 REFERENCES (1) Weiner, G. J. Building better monoclonal antibody-based therapeutics. Nat. Rev. Cancer 2015, 15 (6), 361. (2) Weiner, L. M.; Surana, R.; Wang, S. Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat. Rev. Immunol. 2010, 10 (5), 317. (3) Beck, A.; Sanglier-Cianferani, S.; Van Dorsselaer, A. Biosimilar, biobetter, and next generation antibody characterization by mass spectrometry. Anal. Chem. 2012, 84 (11), 4637. (4) Reichert, J. M. Antibodies to watch in 2014. MAbs 2014, 6 (1), 5. (5) Duong, M. N.; Cleret, A.; Matera, E. L.; Chettab, K.; Mathe, D.; Valsesia-Wittmann, S.; Clemenceau, B.; Dumontet, C. Adipose cells promote resistance of breast cancer cells to trastuzumab-mediated antibody-dependent cellular cytotoxicity. Breast Cancer Res. 2015, 17, 57. (6) Motta, G.; Cea, M.; Moran, E.; Carbone, F.; Augusti, V.; Patrone, F.; Nencioni, A. Monoclonal antibodies for non-Hodgkin's lymphoma: state of the art and perspectives. Clin. Dev. Immunol. 2010, 2010, 428253. (7) Saltz, L.; Easley, C.; Kirkpatrick, P. Panitumumab. Nat. Rev. Drug Discov. 2006, 5 (12), 987. (8) Rathore, A. S.; Winkle, H. Quality by design for biopharmaceuticals. Nat. Biotechnol. 2009, 27 (1), 26. (9) Beck, A.; Wagner-Rousset, E.; Ayoub, D.; Van Dorsselaer, A.; Sanglier-Cianferani, S. Characterization of therapeutic antibodies and related products. Anal. Chem. 2013, 85 (2), 715. (10) Rosati, S.; van den Bremer, E. T.; Schuurman, J.; Parren, P. W.; Kamerling, J. P.; Heck, A. J. In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap. MAbs 2013, 5 (6), 917. (11) Yan, B.; Valliere-Douglass, J.; Brady, L.; Steen, S.; Han, M.; Pace, D.; Elliott, S.; Yates, Z.; Han, Y.; Balland, A.et al. Analysis of post-translational modifications in recombinant monoclonal antibody IgG1 by reversed-phase liquid chromatography/mass spectrometry. J. Chromatogr. A 2007, 1164 (1-2), 153. (12) Zhang, Z.; Pan, H.; Chen, X. Mass spectrometry for structural characterization of therapeutic antibodies. Mass Spectrom. Rev. 2009, 28 (1), 147. 106 (13) Rosati, S.; Yang, Y.; Barendregt, A.; Heck, A. J. Detailed mass analysis of structural heterogeneity in monoclonal antibodies using native mass spectrometry. Nat. Protoc. 2014, 9 (4), 967. (14) Zhang, H.; Cui, W.; Gross, M. L. Mass spectrometry for the biophysical characterization of therapeutic monoclonal antibodies. FEBS Lett. 2014, 588 (2), 308. (15) Song, T.; Ozcan, S.; Becker, A.; Lebrilla, C. B. In-depth method for the characterization of glycosylation in manufactured recombinant monoclonal antibody drugs. Anal. Chem. 2014, 86 (12), 5661. (16) Cai, B.; Pan, H.; Flynn, G. C. C-terminal lysine processing of human immunoglobulin G2 heavy chain in vivo. Biotechnol. Bioeng. 2011, 108 (2), 404. (17) Liu, Y. D.; van Enk, J. Z.; Flynn, G. C. Human antibody Fc deamidation in vivo. Biologicals 2009, 37 (5), 313. (18) Montesino, R.; Calvo, L.; Vallin, A.; Rudd, P. M.; Harvey, D. J.; Cremata, J. A. Structural characterization of N-linked oligosaccharides on monoclonal antibody Nimotuzumab through process development. Biologicals 2012, 40 (4), 288. (19) Maeda, E.; Kita, S.; Kinoshita, M.; Urakami, K.; Hayakawa, T.; Kakehi, K. Analysis of nonhuman N-glycans as the minor constituents in recombinant monoclonal antibody pharmaceuticals. Anal. Chem. 2012, 84 (5), 2373. (20) Huang, L.; Lu, J.; Wroblewski, V. J.; Beals, J. M.; Riggin, R. M. In vivo deamidation characterization of monoclonal antibody by LC/MS/MS. Anal. Chem. 2005, 77 (5), 1432. Bailey, M. J.; Hooker, A. D.; Adams, C. S.; Zhang, S.; James, D. C. A platform for highthroughput molecular characterization of recombinant monoclonal antibodies. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2005, 826 (1-2), 177. (21) (22) Zhang, Z.; Shah, B. Characterization of variable regions of monoclonal antibodies by topdown mass spectrometry. Anal. Chem. 2007, 79 (15), 5723. (23) Bondarenko, P. V.; Second, T. P.; Zabrouskov, V.; Makarov, A. A.; Zhang, Z. Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-Orbitrap mass spectrometer. J. Am. Soc. Mass. Spectrom. 2009, 20 (8), 1415. (24) Tsybin, Y. O.; Fornelli, L.; Stoermer, C.; Luebeck, M.; Parra, J.; Nallet, S.; Wurm, F. M.; Hartmer, R. Structural analysis of intact monoclonal antibodies by electron transfer dissociation mass spectrometry. Anal. Chem. 2011, 83 (23), 8919. (25) Mao, Y.; Valeja, S. G.; Rouse, J. C.; Hendrickson, C. L.; Marshall, A. G. Top-down structural analysis of an intact monoclonal antibody by electron capture dissociation- 107 Fourier transform ion cyclotron resonance-mass spectrometry. Anal. Chem. 2013, 85 (9), 4239. (26) Srzentic, K.; Fornelli, L.; Laskay, U. A.; Monod, M.; Beck, A.; Ayoub, D.; Tsybin, Y. O. Advantages of extended bottom-up proteomics using Sap9 for analysis of monoclonal antibodies. Anal. Chem. 2014, 86 (19), 9945. (27) Ayoub, D.; Bertaccini, D.; Diemer, H.; Wagner-Rousset, E.; Colas, O.; Cianferani, S.; Van Dorsselaer, A.; Beck, A.; Schaeffer-Reiss, C. Characterization of the N-Terminal Heterogeneities of Monoclonal Antibodies Using In-Gel Charge Derivatization of alphaAmines and LC-MS/MS. Anal. Chem. 2015, 87 (7), 3784. (28) Li, H.; Ortiz, R.; Tran, L. T.; Salimi-Moosavi, H.; Malella, J.; James, C. A.; Lee, J. W. Simultaneous analysis of multiple monoclonal antibody biotherapeutics by LC-MS/MS method in rat plasma following cassette-dosing. AAPS J. 2013, 15 (2), 337. (29) Zhang, T.; Zhang, J.; Hewitt, D.; Tran, B.; Gao, X.; Qiu, Z. J.; Tejada, M.; GazzanoSantoro, H.; Kao, Y. H. Identification and characterization of buried unpaired cysteines in a recombinant monoclonal IgG1 antibody. Anal. Chem. 2012, 84 (16), 7112. (30) Du, Y.; Wang, F.; May, K.; Xu, W.; Liu, H. LC-MS analysis of glycopeptides of recombinant monoclonal antibodies by a rapid digestion procedure. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2012, 907, 87. (31) Wang, Y.; Lu, Q.; Wu, S. L.; Karger, B. L.; Hancock, W. S. Characterization and comparison of disulfide linkages and scrambling patterns in therapeutic monoclonal antibodies: using LC-MS with electron transfer dissociation. Anal. Chem. 2011, 83 (8), 3133. (32) Lesur, A.; Varesio, E.; Hopfgartner, G. Accelerated tryptic digestion for the analysis of biopharmaceutical monoclonal antibodies in plasma by liquid chromatography with tandem mass spectrometric detection. J. Chromatogr. A 2010, 1217 (1), 57. (33) Xiang, T.; Chumsae, C.; Liu, H. Localization and quantitation of free sulfhydryl in recombinant monoclonal antibodies by differential labeling with 12C and 13C iodoacetic acid and LC-MS analysis. Anal. Chem. 2009, 81 (19), 8101. (34) Rehder, D. S.; Dillon, T. M.; Pipes, G. D.; Bondarenko, P. V. Reversed-phase liquid chromatography/mass spectrometry analysis of reduced monoclonal antibodies in pharmaceutics. J. Chromatogr. A 2006, 1102 (1-2), 164. (35) Wang, L.; Amphlett, G.; Lambert, J. M.; Blattler, W.; Zhang, W. Structural characterization of a recombinant monoclonal antibody by electrospray time-of-flight mass spectrometry. Pharm. Res. 2005, 22 (8), 1338. 108 (36) Zhang, J.; Liu, H.; Katta, V. Structural characterization of intact antibodies by highresolution LTQ Orbitrap mass spectrometry. J. Mass Spectrom. 2010, 45 (1), 112. (37) Wang, B.; Gucinski, A. C.; Keire, D. A.; Buhse, L. F.; Boyne, M. T., 2nd. Structural comparison of two anti-CD20 monoclonal antibody drug products using middle-down mass spectrometry. Analyst 2013, 138 (10), 3058. (38) Fornelli, L.; Ayoub, D.; Aizikov, K.; Beck, A.; Tsybin, Y. O. Middle-down analysis of monoclonal antibodies with electron transfer dissociation orbitrap fourier transform mass spectrometry. Anal. Chem. 2014, 86 (6), 3005. (39) An, Y.; Zhang, Y.; Mueller, H. M.; Shameem, M.; Chen, X. A new tool for monoclonal antibody analysis: application of IdeS proteolysis in IgG domain-specific characterization. MAbs 2014, 6 (4), 879. (40) Wang, D.; Wynne, C.; Gu, F.; Becker, C.; Zhao, J.; Mueller, H. M.; Li, H.; Shameem, M.; Liu, Y. H. Characterization of drug-product-related impurities and variants of a therapeutic monoclonal antibody by higher energy C-trap dissociation mass spectrometry. Anal. Chem. 2015, 87 (2), 914. (41) Nicolardi, S.; Deelder, A. M.; Palmblad, M.; van der Burgt, Y. E. Structural analysis of an intact monoclonal antibody by online electrochemical reduction of disulfide bonds and Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 2014, 86 (11), 5376. (42) Fornelli, L.; Damoc, E.; Thomas, P. M.; Kelleher, N. L.; Aizikov, K.; Denisov, E.; Makarov, A.; Tsybin, Y. O. Analysis of intact monoclonal antibody IgG1 by electron transfer dissociation Orbitrap FTMS. Mol. Cell. Proteomics. 2012, 11 (12), 1758. (43) Zang, L.; Carlage, T.; Murphy, D.; Frenkel, R.; Bryngelson, P.; Madsen, M.; Lyubarskaya, Y. Residual metals cause variability in methionine oxidation measurements in protein pharmaceuticals using LC-UV/MS peptide mapping. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2012, 895-896, 71. (44) Dick, L. W., Jr.; Mahon, D.; Qiu, D.; Cheng, K. C. Peptide mapping of therapeutic monoclonal antibodies: improvements for increased speed and fewer artifacts. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009, 877 (3), 230. (45) Yang, H.; Zubarev, R. A. Mass spectrometric analysis of asparagine deamidation and aspartate isomerization in polypeptides. Electrophoresis 2010, 31 (11), 1764. (46) Hahne, H.; Pachl, F.; Ruprecht, B.; Maier, S. K.; Klaeger, S.; Helm, D.; Medard, G.; Wilm, M.; Lemeer, S.; Kuster, B. DMSO enhances electrospray response, boosting sensitivity of proteomic experiments. Nat. Methods 2013, 10 (10), 989. 109 (47) Swaney, D. L.; Wenger, C. D.; Coon, J. J. Value of using multiple proteases for largescale mass spectrometry-based proteomics. J. Proteome. Res. 2010, 9 (3), 1323. (48) Wu, C.; Tran, J. C.; Zamdborg, L.; Durbin, K. R.; Li, M.; Ahlf, D. R.; Early, B. P.; Thomas, P. M.; Sweedler, J. V.; Kelleher, N. L. A protease for 'middle-down' proteomics. Nat. Methods 2012, 9 (8), 822. (49) Yuan, Z. F.; Arnaudo, A. M.; Garcia, B. A. Mass spectrometric analysis of histone proteoforms. Annu. Rev. Anal. Chem. 2014, 7, 113. (50) Tan, Y. J.; Wang, W. H.; Zheng, Y.; Dong, J.; Stefano, G.; Brandizzi, F.; Garavito, R. M.; Reid, G. E.; Bruening, M. L. Limited proteolysis via millisecond digestions in proteasemodified membranes. Anal. Chem. 2012, 84 (19), 8357. (51) Good, D. M.; Wirtala, M.; McAlister, G. C.; Coon, J. J. Performance characteristics of electron transfer dissociation mass spectrometry. Mol. Cell. Proteomics 2007, 6 (11), 1942. (52) Han, W.; Yamauchi, M.; Hasegawa, U.; Noda, M.; Fukui, K.; van der Vlies, A. J.; Uchiyama, S.; Uyama, H. Pepsin immobilization on an aldehyde-modified polymethacrylate monolith and its application for protein analysis. J. Biosci. Bioeng. 2015, 119 (5), 505. (53) Long, Y.; Wood, T. D. Immobilized pepsin microreactor for rapid peptide mapping with nanoelectrospray ionization mass spectrometry. J. Am. Soc. Mass. Spectrom. 2015, 26 (1), 194. (54) Udeshi, N. D.; Compton, P. D.; Shabanowitz, J.; Hunt, D. F.; Rose, K. L. Methods for analyzing peptides and proteins on a chromatographic timescale by electron-transfer dissociation mass spectrometry. Nat. Protoc. 2008, 3 (11), 1709. (55) Tan, Y. J.; Sui, D.; Wang, W. H.; Kuo, M. H.; Reid, G. E.; Bruening, M. L. Phosphopeptide enrichment with TiO2-modified membranes and investigation of tau protein phosphorylation. Anal. Chem. 2013, 85 (12), 5699. (56) Beck, A.; Debaene, F.; Diemer, H.; Wagner-Rousset, E.; Colas, O.; Van Dorsselaer, A.; Cianferani, S. Cutting-edge mass spectrometry characterization of originator, biosimilar and biobetter antibodies. J. Mass Spectrom. 2015, 50 (2), 285. (57) Marcoux, J.; Champion, T.; Colas, O.; Wagner-Rousset, E.; Corvaia, N.; Van Dorsselaer, A.; Beck, A.; Cianferani, S. Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate. Protein Sci. 2015, 24, 1210. 110 (58) Lew, C.; Gallegos-Perez, J. L.; Fonslow, B.; Lies, M.; Guttman, A. Rapid level-3 characterization of therapeutic antibodies by capillary electrophoresis electrospray ionization mass spectrometry. J. Chromatogr. Sci. 2015, 53 (3), 443. (59) Gahoual, R.; Burr, A.; Busnel, J. M.; Kuhn, L.; Hammann, P.; Beck, A.; Franccois, Y. N.; Leize-Wagner, E. Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry. Mabs 2013, 5 (3), 479. (60) Xie, H.; Chakraborty, A.; Ahn, J.; Yu, Y. Q.; Dakshinamoorthy, D. P.; Gilar, M.; Chen, W.; Skilton, S. J.; Mazzeo, J. R. Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies. MAbs 2010, 2 (4), 379. (61) Olsen, J. V.; Macek, B.; Lange, O.; Makarov, A.; Horning, S.; Mann, M. Higher-energy C-trap dissociation for peptide modification analysis. Nat. Methods 2007, 4 (9), 709. (62) Hu, D.; Qin, Z.; Xue, B.; Fink, A. L.; Uversky, V. N. Effect of methionine oxidation on the structural properties, conformational stability, and aggregation of immunoglobulin light chain LEN. Biochemistry 2008, 47 (33), 8665. 111 Chapter 3 . Enzyme-Containing Spin Membranes for Rapid Protein Digestion (Reproduced with permission from Analytical Chemistry, submitted for publication. Unpublished work copyright (2017) American Chemical Society.) Proteolytic digestion is an important step in protein characterization using mass spectrometry (MS). This study uses pepsin- or trypsin-containing spin membranes for rapid protein digestion prior to ultrahigh-resolution Orbitrap MS analysis. Centrifugation of 100 ÂľL of pretreated protein solutions through the functionalized membranes requires less than 1 min. Peptic and tryptic peptides from spin digestion of apomyoglobin and four commercial monoclonal antibodies (mAbs) cover nearly 100% of the protein sequences in direct infusion MS analysis. Increasing the spin rate leads to a higher fraction of large peptic peptides for apomyoglobin, and MS analysis of peptic and tryptic peptides reveals post-translational mAb modifications such as Nterminal pyroglutamate formation, C-terminal Lysine clipping and glycosylation. Analysis of tryptic spin digests with liquid chromatography coupled to tandem mass spectrometry (LCMS/MS) and MaxQuant data searching gives 100% sequence coverage of all four antibody light chains, and 75.1%-98.4% coverage of the heavy chains. Compared to in-solution tryptic digestion of mAbs, trypsin spin digestion yields higher sequence coverage and a larger number of unique peptides. 3.1 Introduction Proteolysis is often a crucial step in protein characterization, identification, and quantitation through mass spectrometry (MS) and/or tandem mass spectrometry (MS/MS) analysis.1 112 Compared to MS and MS/MS characterization of intact proteins, analyses of proteolytic peptides yield greater sequence information as well as greater resolution in separations with liquid chromatography (LC).2 However, conventional peptide generation using in-solution digestion requires long incubation times (up to 24 h) because of the low enzyme concentrations required to avoid self-digestion.3-7 Unfortunately, oxidation or other protein modifications may occur during long digestions.8,9 Minimizing hydrogen/deuterium exchange during digestion is also vital to avoid back exchange in studies of protein structure based on hydrogen/deuterium exchange prior to digestion.10 Thus, rapid digestion is important for a number of protein characterization studies. Several research groups and companies developed immobilized-enzyme reactors for rapid protein digestion.11,12 The high enzyme-to-protein ratio in these reactors greatly improves the digestion rate, and immobilization can also increase enzyme stability and decrease autolysis.3-7,13 Solid supports employed to create immobilized-enzyme reactors include monoliths,14-29 capillaries,30,31 magnetic particles,32-34 resins,35-38 microfluidic chips,39,40 and membranes.41-44 We are particularly interested in membrane supports because they are inexpensive, and varying the flowrate through these thin structures affords solution residence times that range from msec to sec. Initially, enzyme immobilization in membranes relied on hydrophobic interactions in poly (vinylidene difluoride).44,45 Xu and coworkers later formed a trypsin-containing membrane through sequential adsorption of poly (styrene sulfonate) (PSS) and trypsin in porous nylon.41 PSS absorbs strongly to nylon, presumably through multiple hydrophobic interactions, to create a negatively charged surface. With a pI of ~10.5, trypsin is positively charged in acidic solution and electrostatically adsorbs to negatively charged PSS-modified membranes. This adsorption procedure gives a membrane reactor with a local concentration of 10 mg of trypsin per mL of 113 membrane pores, which is 450 times higher than the typical trypsin concentration for in-solution digestion. The short radial diffusion distances within the microporous membrane pores further facilitate rapid digestion. Tan et al. used a similar strategy to form pepsin-containing membranes.42 Recently, we exploited a pepsin-containing membrane to facilitate monoclonal antibody (mAb) characterization.43 By varying the antibody residence time (from 3 ms to 3 s) in the membrane, we obtained “bottom-up” (1-2 kDa) to “middle-down” (5-15 kDa) sized peptides, and these peptides cover the entire sequences of two different antibodies. However, for all the aforementioned membrane-based protein digestions, protein passage through the membrane employed relatively cumbersome systems that included syringe or peristaltic pumps. To overcome this challenge, we recently developed a membrane fitting that attaches to a disposable pipette tip.46 This allows rapid digestion, but loading the membrane into the ferrule fitting, achieving a good seal, and extended production of the device are challenging. This chapter describes protein digestion using spin membranes containing immobilized pepsin or trypsin. During centrifugation, protein solutions pass through the spin membrane in 1 min or less to yield proteolytic peptides for subsequent direct infusion or LC-MS/MS analysis with an Orbitrap ultrahigh resolution mass spectrometer. Direct infusion analysis of apomyoglobin and four commercial monoclonal antibodies (Herceptin, Avastin, Rituxan and Vectibix) yields nearly 100% sequence coverage. LC-MS/MS analysis of tryptic spin digests of four antibodies followed by MaxQuant data processing easily identifies the four antibodies via comparison with a protein database. In these LC-MS/MS analyses, sequence coverages are 100% for all the light chains, and range from 75.1 to 98.4% for the different antibody heavy chains. 114 3.2 Experimental 3.2.1 Materials Nylon membranes (LoProdyne LP, nominal pore size 1.2 Îźm, 110 Îźm thickness) were purchased from Pall Corporation. Trastuzumab (Herceptin, Genentech), Bevacizumab (Avastin, Genentech), Rituxan (Rituximab, Genentech) and Vectibix (Panitumumab, Amgen) were obtained in their commercial formulations as a gift from Dr. Muhammad Chisti of Michigan State University. Trypsin from bovine pancreas (TPCK-treated, lyophilized powder, ≥10,000 BAEE units/mg protein), pepsin from porcine gastric mucosa (lyophilized powder, 3200-4500 units/mg protein), ammonium bicarbonate (≥99%), iodoacetamide (IAM, ≥99%), dithiothreitol (DTT, ≥99.5%), polystyrene sulfonate (PSS, average molecular weight ~70,000), formic acid (FA, ≥98%) and acetonitrile (ACN, HPLC grade, ≥99.9%) were purchased from Sigma Aldrich. Sequencing grade modified trypsin was obtained from Promega. NaCl (ACS grade) and HCl (ACS grade) were purchased from CCI. Other chemicals include urea ( ≥ 98%, Invitrogen), tris(2carboxyethyl) phosphine hydrochloride (TCEP-HCl, >98%, Fluka), trifluoroacetic acid (TFA, EMD), acetic acid (HOAc, ACS, Macron Fine Chemicals), and methyl alcohol (anhydrous, MeOH, Macron Fine Chemicals). Solutions were prepared in deionized water (DI water, Milli-Q, 18.2 MΊ¡cm at 25 °C). C4 ZipTips were purchased from EMD Millipore, and Pierce C18 spin columns were used to isolate tryptic peptides after digestion. Amicon ultra 0.5 mL centrifugal filters (MWCO 10 kDa) were employed to desalt samples before pepsin in-membrane digestion, and an Eppendorf centrifuge (5415D) was used to conduct spin digestion. 115 3.2.2 Functionalized Membrane-Containing Spin Columns Trypsin- and pepsin-containing membranes were prepared using a slight modification of our literature procedure.41-43 Membranes were UV/ozone-cleaned for 10 min, and 10 mL of 0.02 M PSS in 0.5 M NaCl (pH=2.3) was circulated through the membrane for 10 min using a peristaltic pump, followed by rinsing with 30 mL of DI water. For trypsin-containing membranes after adsorption of PSS, 5 mL of 1 mg/mL trypsin in 2.7 mM HCl was circulated through the membrane for 1 h. Subsequently, the membrane was rinsed with 30 mL of 1 mM HCl, dried with N2, and stored in a desiccator. For pepsin-containing membranes, 4 mL of 2 mg/mL pepsin in 5% FA was circulated through the membrane for 1 h. Then, the membrane was rinsed with 30 mL of 5% FA, dried with N2, and stored in a desiccator. Flow rates during membrane modification were 2 mL/min. The modified membranes were embedded in spin devices at Takara/Clontech Laboratories (Mountain View, CA). These devices expose a membrane surface with a diameter of ~1.8 mm. 3.2.3 Apomyoglobin spin digestion with pepsin- and trypsin-containing membranes Apomyoglobin (10 Âľg) was dissolved in 100 ÂľL of 10 mM NH4HCO3 for trypsin digestion, and in 100 ÂľL of 5% FA for pepsin digestion. The spin column was rinsed with 100 ÂľL of 10 mM NH4HCO3 or 100 ÂľL of 5% FA before tryptic or peptic spin digestion, respectively. Both enzymatic digestions were conducted at three spin rates corresponding to 500 g and 10,000 g. The centrifugation time was 1 min, and the digests were dried with a SpeedVac after spin digestion and immediately reconstituted for MS analysis. 116 3.2.4 mAb spin digestion with pepsin- and trypsin-containing membranes For pepsin digestion, Trastuzumab, Bevacizumab, Rituximab and Panitumumab were each diluted in deionized water to prepare stock solutions with 1 mg/mL of antibody. Subsequently, 2 ÂľL of 0.1 M HOAc and 2 ÂľL of 0.1 M TCEP-HCl were added to 20 ÂľL of mAb stock solutions, and these mixtures were incubated at 75 °C for 15 min. Subsequent buffer exchange with 5% FA employed 3 cycles of centrifugation with an Amicon ultra 0.5 mL centrifugal filter (MWCO 10 kDa). About 25 ÎźL of solution remained after each centrifugation, and 475 ÎźL of 5% FA was added prior to the following centrifugation. Residues were diluted to 200 ÂľL with 5% FA to make 0.1 mg/mL solutions. For trypsin digestion, 4 ÎźL of 10 mg/mL antibody stock solutions of each of the four mAbs were diluted separately in 14 ÎźL of 2 mM TCEP-HCl solution in 0.1% HOAc containing 8 M urea. The mixtures were incubated at 50 °C for 10 min prior to addition of 14 ÂľL of 20 mM IAM in 2 M NH4HCO3 containing 8 M urea, and incubation in the dark for 30 min. Finally, 12 ÂľL of 30 mM DTT in 100 mM NH4HCO3 containing 8 M urea was added followed by incubation in the dark for 20 min to quench the IAM. After reduction and alkylation, the residual solutions were diluted with deionized water to create 0.1 mg/mL solutions. 3.2.4.1 In-membrane spin digestion of mAbs Within 1.5 h of antibody pretreatments, 200 ÂľL of each nonalkylated antibody solution was added to a pepsin spin column, and 200 ÂľL of each alkylated antibody solution was added to a trypsin spin column. The solutions were centrifuged through the membrane for 1 min at 500 g. Pepsin spin digestion samples were collected for direct infusion MS and LC-MS/MS analysis, whereas trypsin spin digestion samples were first desalted using Pierce C18 spin cartridges 117 (following the manufacturer’s protocol) before infusion and LC/MS analysis. The C18 spin column was activated with 50% MeOH, and equilibrated in 0.5% TFA in 5% ACN. Then, the sample was loaded onto the column, followed by washing with of 0.5% TFA in 5% ACN. Finally, the peptides were eluted from the spin column with 70% ACN. Antibody in-membrane spin digestions were also monitored by SDS-PAGE. The reproducibility of the spin-membrane digestion was tested by running triplicate trypsin and pepsin digestions of Bevacizumab with a separate membrane for each digestion. 3.2.4.2 In-solution trypsin digestion of mAbs We conducted in-solution tryptic digestion of four antibodies to compare in-membrane and spin digestion. Five microliters of 0.2 Îźg/ÎźL sequencing grade modified trypsin solution was added to 200 ÎźL of the 0.1 mg/mL alkylated antibody solution prior to incubation at 37 °C for 16 h. The reaction was quenched by adding 5 ÎźL of acetic acid. Samples were then desalted with a C18 spin column and dried with a SpeedVac before reconstitution and infusion MS or LC-MS/MS analysis. 3.2.5 Mass Spectrometry and Data Analysis For direct infusion MS, in-membrane spin digests and in-solution digests were dried with a SpeedVac and reconstituted in 1% acetic acid, 49% H2O, and 50% methanol within 1 day. Then, 40 ÎźL of each sample was loaded into a Whatman multichem 96-well plate (Sigma−Aldrich) and sealed with Teflon Ultrathin Sealing Tape (Analytical Sales and Services, Prompton Plains, NJ). An Advion Triversa Nanomate nanoelectrospray ionization (nESI) source (Advion, Ithaca, NY) was used to introduce the sample into a high-resolution accurate mass Thermo Fisher Scientific LTQ Orbitrap Velos mass spectrometer (San Jose, CA) that was equipped with a dual pressure 118 ion trap, HCD cell, and ETD. The spray voltage and gas pressure were set to 1.4 kV and 1.0 psi, respectively. The ion-source interface had an inlet temperature of 200 °C with an S-Lens value of 57%. High-resolution mass spectra were acquired in positive ionization mode across the m/z range of 400−1800, using the FT analyzer operating at a mass resolving power of 100,000. Spectra were the average of 100 scans. Signals with >1% of the highest peak intensities and S/N>3 were analyzed. Peptide identification was performed manually using ProteinProspector (v 5.14.1, University of California, San Francisco, CA). Mass tolerance was set to 10 ppm. For LC-MS/MS, Nano-Ultra High Performance Liquid Chromatography MS/MS was performed essentially as described previously.47 Briefly, 2uL injections corresponding to 500 ng of spindigested tryptic protein (reconstituted in 0.1% FA) were loaded onto a 100 mm x 75 um C18BEH column (Waters Billerica, MA), and separated over a 90 min gradient from 5-35%B on a nano-Acquity system (Waters) flowing at 500 nL/min. Solution A was 0.1% FA in H2O, and solution B was 0.1% FA in ACN. MS/MS was performed on an LTQ-Velos Orbitrap-FTMS (Thermo, San Jose, CA) running a top-20 data dependent method, where a single MS at a resolution of 60,000 was acquired, and the top-20 precursors were selected for fragmentation. Raw LC-MS/MS files were processed by MaxQuant version 1.5.6.0. MS/MS spectra were searched against the Cricetulus griseus (Chinese hamster) proteome (23,884 proteins). The database also included common contaminants and the antibody sequences. MaxQuant analysis parameters included a precursor mass tolerance of 20 ppm for the initial search, a precursor mass tolerance of 6 ppm for the main search, and an FTMS MS/MS match tolerance of 20 ppm. We set trypsin as the specific enzyme. Variable modifications included oxidation (M), deamidation (NQ), and Gln->pyro-Glu, while the fixed modification was carbamidomethyl on cysteine. The 119 minimal peptide length was set to 6 amino acids, the maximum peptide mass was 8000 Da, and the maximum number of missed cleavages was 5. 3.3 Results and discussion 3.3.1 Workflow for Digestion in Membrane-Containing Spin Columns Figure 3.1 shows the workflow that we use to conduct protein digestion in spin membranes. After protein pretreatment, the solution simply passes through the membrane reactor during centrifugation. Digestion of 100 ÎźL of protein solution requires a centrifugation time less than 30 sec. The high concentration of enzyme in the membrane pores affords rapid digestion of protein, and we can control the digestion by varying the spin rate. Moreover, the tiny dead volume (0.275 ÎźL) of the spin membrane should minimize peptide loss during digestion. Figure 3.1. Workflow for protein spin digestion and analysis. [Acronyms: VH-variable region of the heavy chain; CH1, CH2, and CH3- different constant regions of the heavy chain; CLconstant region of the light chain; VL- variable region of the light chain; LC- light chain; HCheavy chain.] 120 3.3.2 Apomyoglobin spin digestion with pepsin- and trypsin-containing membranes We chose apomyoglobin, a common standard for peptide mapping, to initially test the spin digestion. Apomyoglobin has a compact hydrophobic core at neutral pH and undergoes slow insolution proteolysis at pH 8.48 Reduction and alkylation are not necessary for digesting this protein because it has no disulfide bonds. Using both trypsin and pepsin spin membranes and different spin rates prior to infusion MS analysis, we always observed 100% apomyoglobin sequence coverage (percent of protein sequence covered by identified proteolytic peptides) after spin digestion in a single pass through the membrane. 121 Figure 3.2. Deconvoluted ESI-Orbitrap mass spectra of apomyoglobin peptic digests obtained through 500 g (top) and 10,000 g (bottom) spin digestion. Deconvoluted mass spec were generated with Xtract software. Figure 3.2 shows ESI-Orbitrap deconvoluted mass spectra of peptic apomyoglobin digests obtained using spin digestion at 500 g and 10,000 g. Four peptides, 1-29, 30-106, 107-137, and 138-153 cover the whole sequence after digestion at 500 g, whereas three peptides 1-29, 30-106, and 107-153 do the same thing after digestion at 10,000 g. When the spin rate increases to 10,000 g, the signals of large peptides such as amino acids 30-106 and 107-153 increase dramatically. At the same time, signals for smaller peptides, including amino acids 30-69 and 70- 122 106 decrease. Increasing the spin rate leads to a higher fraction of large peptides, presumably because lower residence times in the membrane decrease the proteolysis time to generate more missed cleavages. By varying the centrifugation rate, we can obtain overlapping peptides using a single enzyme. The aspartic protease pepsin exhibits less specificity than trypsin.49 However, extensive studies of pepsin digestion show that this protease prefers to cleave peptide bonds after phenylalanine (F) and leucine (L).50 Our results match the cleavage site preferences for pepsin. Peptic peptides 3069, 70-106, 107-153, and 138-153 result from cleavage of the 29L-30I, 69L-70T, 106F-107I, and 137L-138F bonds. Table 3.1 and Table 3.2 show the peptic peptides identified from digestion at 500 g and 10,000 g, respectively. Trypsin is the most common enzyme used for protein digestion, particularly for bottom-up proteomics.51 Compared with pepsin, it has higher specificity, cleaving proteins and peptides at the C-terminus of K and R, except when followed by P. Moreover, at low pH tryptic peptides carry at least two positive charges, which benefits downstream collision-induced dissociation tandem mass spectrometry (CID-MS/MS) analysis. Figure 4.3 shows the deconvoluted ESIOrbitrap mass spectrum of a tryptic spin digest of apomyoglobin. A spin at 500 g gives complete digestion of apomyoglobin (no intact apomyoglobin) in one pass through the membrane. We identified 26 tryptic peptides, and as few as seven tryptic peptides cover 100% of the sequence: amino acids 1-31, 32-47, 48-63, 64-77, 78-96, 97-133, and 134-153. Table 3.3 gives a full list of the identified peptides, most of which contain 1 or more missed cleavage sites. Different from peptic spin digestion, we don’t see the emergence of large peptides at higher rates of centrifugation. Instead we see signals of undigested, intact protein. This may stem from the compact structure of apomyoglobin at pH 7-8 as well as the high activity of trypsin. After an 123 initial cleavage, structures of the resulting peptides likely open rapidly to allow further digestion, although the initial cleavage is slow. Thus, changing the spin rate yields intact protein rather than limited proteolysis. In future studies, covalent linking of trypsin to the membrane may decrease its activity to better obtain limited proteolysis. Figure 3.3. Part of the mass spectrum of a tryptic spin digests (500 g) of apomyoglobin. Labels show the amino acid (not all of the peptide signals are labelled). 124 Table 3.1. Apomyoglobin peptides identified from a spin-membrane (spun at 500 g) peptic digest. m/z of [M+H]+ 3134.5542 4651.5069 8764.8436 4132.3546 3242.6627 2927.5196 1856.9654 Peptide Sequence (-)GLSDGEWQQVLNVWGKVEADIAGHGQE VL(I) (L)IRLFTGHPETLEKFDKFKHLKTEAEMKASEDL KKHGTVVL(T) (L)IRLFTGHPETLEKFDKFKHLKTEAEMKASEDL KKHGTVVLTALGGILKKKGHHEAELKPLAQSHA TKHKIPIKYLEF(I) (L)TALGGILKKKGHHEAELKPLAQSHATKHKIPI KYLEF(I) (F)ISDAIIHVLHSKHPGDFGADAQGAMTKALEL(F ) (D)AIIHVLHSKHPGDFGADAQGAMTKALE L(F) (L)FRNDIAAKYKELGFQG(-) Amino Acids 1-29 30-69 30-106 70-106 107-137 110-137 138-153 Table 3.2. Apomyoglobin peptides identified from a spin-membrane (spun at 10,000 g) peptic digest. m/z of [M+H]+ 3134.5542 4651.5069 8764.8436 9079.9867 11988.4885 4132.3546 3242.6627 5080.6102 2927.5196 1856.9654 Peptide Sequence (-)GLSDGEWQQVLNVWGKVEADIAGHGQE VL(I) (L)IRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKK HGTVVL(T) (L)IRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKK HGTVVLTALGGILKKKGHHEAELKPLAQSHATKHK IPIKYLEF(I) (L)IRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKK HGTVVLTALGGILKKKGHHEAELKPLAQSHATKHK IPIKYLEFISD(A) (L)IRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKK HGTVVLTALGGILKKKGHHEAELKPLAQSHATKHK IPIKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKAL EL(F) (L)TALGGILKKKGHHEAELKPLAQSHATKHKIPIKY LEF(I) (F)ISDAIIHVLHSKHPGDFGADAQGAMTKALEL(F) (F)ISDAIIHVLHSKHPGDFGADAQGAMTKALELFRN DIAAKYKELGFQG(-) (D)AIIHVLHSKHPGDFGADAQGAMTKALE L(F) (L)FRNDIAAKYKELGFQG(-) 125 Amino Acids 1-29 30-69 30-106 30-109 30-137 70-106 107-137 107-153 110-137 138-153 Table 3.3. Apomyoglobin peptides identified from a spin-membrane (spun at 500 g) tryptic digest. m/z of [M+H]+ 1815.9024 3403.7393 1606.8547 1271.663 1937.0167 3004.5601 1086.5612 1857.9739 790.4305 1378.8417 1506.9366 1635.0316 2110.1516 1982.0566 1853.9617 735.4876 2601.4915 4085.143 3819.9891 1885.0218 3368.6732 1502.6693 1360.7583 2283.2132 922.4993 941.4727 Peptide Sequence (-)GLSDGEWQQVLNVWGK(V) (-)GLSDGEWQQVLNVWGKVEADIAGHGQE VLIR(L) (K)VEADIAGHGQEVLIR(L) (R)LFTGHPETLEK(F) (R)LFTGHPETLEKFDKFK(H) (R)LFTGHPETLEKFDKFKHLKTEAEMK(A) (K)HLKTEAEMK(A) (K)HLKTEAEMKASEDLKK(H) (K)ASEDLKK(H) (K)HGTVVLTALGGILK(K) (K)HGTVVLTALGGILKK(K) (K)HGTVVLTALGGILKKK(G) (K)KKGHHEAELKPLAQSHATK(H) (K)KGHHEAELKPLAQSHATK(H) (K)GHHEAELKPLAQSHATK(H) (K)HKIPIK(Y) (K)HKIPIKYLEFISDAIIHVLHSK(H) (K)HKIPIKYLEFISDAIIHVLHSKHPGDFGADAQG AMTK(A) (K)IPIKYLEFISDAIIHVLHSKHPGDFGADAQGAM TK(A) (K)YLEFISDAIIHVLHSK(H) (K)YLEFISDAIIHVLHSKHPGDFGADAQGAMTK( A) (K)HPGDFGADAQGAMTK(A) (K)ALELFRNDIAAK(Y) (K)ALELFRNDIAAKYKELGFQG(-) (R)NDIAAKYK(E) (K)YKELGFQG(-) Amino Acids 1-16 1-31 17-31 32-42 32-47 32-56 48-56 48-63 57-63 64-77 64-78 64-79 78-96 79-96 80-96 97-102 97-118 97-133 99-133 103-118 103-133 119-133 134-145 134-153 140-147 146-153 3.3.3 mAb spin digestion with pepsin-containing membranes Antibodies have unique Y-shape structures that include inter- and intra-chain disulfide bonds.52 Thus, we used mAbs as model proteins with disulfide bonds. Moreover, enzymatic digestion is crucial for antibody characterization, structure analysis and quality control. We previously reported using a cumbersome homemade setup with a syringe pump for controlled antibody in- 126 membrane peptic digestion.43 Here we examine spin digestion using both trypsin- and pepsincontaining membranes of four different therapeutic antibodies. Proper antibody pretreatment, which is vital to effective, reproducible digestion, is different for pepsin and trypsin. For peptic digestion, we used TCEP as the reducing agent. Unlike DTT, TCEP can reduce disulfide bonds under acidic conditions where pepsin has maximum activity. Moreover, antibodies partially denature at pH 2, and this should increase access to cleavage sites. Acidic conditions also prevent reformation of disulfide bonds after reduction and avoid the need for adding chaotropic and alkylation agents. Different from our previous workflow, just prior to the spin digestion we added a buffer exchange step using 10 kDa molecular weight cutoff membranes. Because of the high concentration of salt in the commercial antibody formulation,53 desalting is important for downstream MS or LC/MS analysis. In digestion, we employed 30 s for spinning 100 ÂľL of desalted antibody solutions through membranes at 500 g, but the time required for the solution to pass through the membrane is actually less than 30 s. Direct-infusion MS analysis of peptic peptides from Herceptin (He), Avastin (Av), Rituxan (Ri) and Vectibix (Ve) gives 100% sequence coverage for all of the antibodies. Figure 3.4 is the sequence map for Avastin, and Figure 3.5-3.7 give sequence maps for the other antibodies. 127 Figure 3.4. Sequence map of the peptides identified from infusion ESI-Orbitrap analysis of peptic digest of Avastin. Light green color “N” represents the glycosylation site. Red color “K” represents the C-terminal clipping. 128 Figure 3.5. Sequence map of the peptides identified from infusion ESI-Orbitrap analysis of peptic digest of Herceptin. Light green color “N” represents the glycosylation site. 129 Figure 3.6. Sequence map of the peptides identified from infusion ESI-Orbitrap analysis of peptic digest of Rituxan. Light green color “N” represents the glycosylation site. Red color “K” represents the C-terminal clipping. 130 Figure 3.7. Sequence map of the peptides identified from infusion ESI-Orbitrap analysis of peptic digest of Vectibix. Light green color “N” represents the glycosylation site. Red color “K” represents the C-terminal clipping. 131 The average length of the 365 identified peptic peptides from He, Av, Ri and Ve spin digestions is 36 amino acids, indicating that spin digestion at 500 g generates middle-down sized peptides that will enable rapid identification of protein posttranslational modifications (PTMs). We identified glycosylation on the heavy chains of all four antibodies. N-terminal pyroglutamate formation appeared on the Ri light chain (Lc), Ri heavy chain (Hc) and Ve Hc. We also saw Cterminal Lysine clipping on Av, Ri and Ve. Figures 3.8-3.11 present the original mass spectra of the four mAbs, and Tables 3.4-3.7 list the peptic peptides identified for the four antibodies. Some signals with the same m/z value are present in the spectra of all four antibodies. For example, signals corresponding to M+H of 8858.2650 result from light chain 136-214 (L136-214) of He, L136-214 of Av, L135-213 of Ri, and L136-214 of Ve. Another peptide with M+H of 4823.3821 stems from heavy chain 408-449 (H408-449) of He, H411-452 of Av, H409-450 of Ri, and H403-444 of Ve. More examples appear in the peptide list in Table 3.4-3.7, as we would expect because the antibodies He, Av, Ri and Ve share the same sequences in a large part of the light-chain and heavy-chain constant regions. The presence of the same peptides in spectra of four antibodies shows that rapid spin digestion is a powerful method for comparing proteins with similar sequences (see chapter 4). Signals that are present in one mass spectrum but not another give hints for the parts that are different in two proteins. 132 Figure 3.8. Part of the mass spectrum of a peptic spin digest of Herceptin. Labels show the amino acids on the Hc and Lc. (not all of the peptide signals are labelled). 133 Figure 3.8 (cont’d) 134 Figure 3.8 (cont’d) 135 Figure 3.9. Part of the mass spectrum of a peptic spin digest of Avastin. Labels show the amino acids on the Hc and Lc. (not all of the peptide signals are labelled). 136 Figure 3.9 (cont’d) 137 Figure 3.9 (cont’d) 138 Figure 3.10. Part of the mass spectrum of a peptic spin digest of Rituxan. Labels show the amino acids on the Hc and Lc. (not all of the peptide signals are labelled). 139 Figure 3.10 (cont’d) 140 Figure 3.10 (cont’d) 141 Figure 3.11. Part of the mass spectrum of a peptic spin digest of Vectibix. Labels show the amino acids on the Hc and Lc. (not all of the peptide signals are labelled). 142 Figure 3.11 (cont’d) 143 Figure 3.11 (cont’d) 144 Table 3.4. Light- and heavy-chain peptides identified from a spin-membrane (spun at 500 g) peptic digest of Herceptin. m/z of [M+H]+ Peptide Sequence 1206.5671 3267.558 (-)DIQMTQSPSSL(S) (-)DIQMTQSPSSLSASVGDRVTITCRASQDVNT(A) (-)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVA W(Y) (-)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVA WYQQKPGKAPKL(L) (-)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVA WYQQKPGKAPKLLIYSAS(F) (-)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVA WYQQKPGKAPKLLIYSASF(L) (-)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVA WYQQKPGKAPKLLIYSASFL(Y) (L)SASVGDRVTITCRASQDVNT(A) (L)SASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKL(L ) (T)AVAWYQQKPGKAPKL(L) (T)AVAWYQQKPGKAPKLLIYSASF(L) (T)AVAWYQQKPGKAPKLLIYSASFL(Y) (W)YQQKPGKAPKLLIYSASF(L) (W)YQQKPGKAPKLLIYSASFL(Y) (F)LYSGVPSRFSGSRSGTD(F) (F)LYSGVPSRFSGSRSGTDF(T) (F)LYSGVPSRFSGSRSGTDFTLTISSLQPED(F) (F)LYSGVPSRFSGSRSGTDFTLTISSLQPEDF(A) (F)LYSGVPSRFSGSRSGTDFTLTISSLQPEDFAT Y(Y) (L)YSGVPSRFSGSRSGTD(F) (L)YSGVPSRFSGSRSGTDF(T) (L)YSGVPSRFSGSRSGTDFTLTISSLQPED(F) (L)YSGVPSRFSGSRSGTDFTLTISSLQPEDF(A) (L)YSGVPSRFSGSRSGTDFTLTISSLQPEDFATY(Y) (F)ATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSV F(I) (F)ATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASV(V) (F)ATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCL(L) (T)YYCQQHYTTPPTFGQGTKVEIKRTVAAPSVF(I) (T)YYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASV(V) (T)YYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCL(L) (Y)YCQQHYTTPPTFGQGTKVEIKRTVAAPSV(F) 3694.7799 4933.4935 5567.8261 5714.8945 5827.9786 2080.0088 3745.9443 1684.9533 2466.3544 2579.4384 2039.1324 2152.2165 1772.8562 1919.9246 3104.5171 3251.5855 3586.7336 1659.7721 1806.8406 2991.433 3138.5014 3473.6496 3689.8421 5346.678 5661.8396 3517.7573 5174.5932 5489.7549 3207.6255 145 Amino Acids L1-11 L1-31 L1-35 L1-46 L1-52 L1-53 L1-54 L12-31 L12-46 L32-46 L32-53 L32-54 L36-53 L36-54 L54-70 L54-71 L54-82 L54-83 L54-86 L55-70 L55-71 L55-82 L55-83 L55-86 L84-116 L84-132 L84-135 L86-116 L86-132 L86-135 L87-115 Table 3.4 (cont’d) 3354.694 5011.5299 5326.6915 3191.6306 1822.9222 2138.0838 1675.8537 1991.0154 7145.4419 9173.4266 6830.2803 8858.265 2047.0025 2256.1653 2618.3243 5982.0872 8678.4066 9050.5897 10107.1097 4775.3576 7074.5207 6813.4423 2225.1826 2411.2619 3382.7807 6079.1002 6451.2833 (Y)YCQQHYTTPPTFGQGTKVEIKRTVAAPSVF(I) (Y)YCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASV(V) (Y)YCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCL(L) (Y)CQQHYTTPPTFGQGTKVEIKRTVAAPSVF(I) (V)FIFPPSDEQLKSGTASV(V) (V)FIFPPSDEQLKSGTASVVCL(L) (F)IFPPSDEQLKSGTASV(V) (F)IFPPSDEQLKSGTASVVCL(L) (V)VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACE(V) (V)VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC(-) (L)LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACE(V) (L)LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC(-) (E)VTHQGLSSPVTKSFNRGEC(-) (-)EVQLVESGGGLVQPGGSLRLSCA(A) (-)EVQLVESGGGLVQPGGSLRLSCAASGF(N) (-)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYI HWVRQAPGKGLEWVARIYPTNG(Y) (-)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYI HWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISA DTSKNTAY(L) (-)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYI HWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISA DTSKNTAYLQM(N) (-)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYI HWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISA DTSKNTAYLQMNSLRAEDTAV(Y) (L)SCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGY TRYAD(S) (L)SCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGY TRYADSVKGRFTISADTSKNTAYLQM(N) (A)ASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY ADSVKGRFTISADTSKNTAYLQM(N) (F)NIKDTYIHWVRQAPGKGLE(W) (F)NIKDTYIHWVRQAPGKGLEW(V) (F)NIKDTYIHWVRQAPGKGLEWVARIYPTNG(Y) (F)NIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSV KGRFTISADTSKNTAY(L) (F)NIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSV KGRFTISADTSKNTAYLQM(N) 146 L87-116 L87-132 L87-135 L88-116 L116-132 L116-135 L117-132 L117-135 L133-195 L133-214 L136-195 L136-214 L196-214 H1-23 H1-27 H1-56 H1-80 H1-83 H1-93 H21-62 H21-83 H24-83 H28-46 H28-47 H28-56 H28-80 H28-83 Table 3.4 (cont’d) 7507.8033 4245.1186 3087.5204 1447.721 2367.0459 1075.5378 1401.6645 1310.5259 1147.4626 984.3992 1304.5616 2952.4983 3065.5823 6637.3811 3118.5884 3703.9006 3590.8166 1812.9226 5914.9122 6425.1924 6572.2608 7708.9202 7839.9607 4631.2877 1944.0663 1433.7861 1286.7177 2273.2032 1136.5438 1005.5034 1104.5718 1898.9283 6705.1696 6867.2114 (F)NIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSV KGRFTISADTSKNTAYLQMNSLRAEDTAV(Y) (E)WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQM (N) (G)YTRYADSVKGRFTISADTSKNTAYLQM(N) (Y)LQMNSLRAEDTAV(Y) (M)NSLRAEDTAVYYCSRWGGDGF(Y) (M)NSLRAEDTAV(Y) (M)NSLRAEDTAVYY(C) (V)YYCSRWGGDGF(Y) (Y)YCSRWGGDGF(Y) (Y)CSRWGGDGF(Y) (F)YAMDYWGQGTL(V) (L)VTVSSASTKGPSVFPLAPSSKSTSGGTAALGC(L) (L)VTVSSASTKGPSVFPLAPSSKSTSGGTAALGC L(V) (L)VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGL(Y) (C)LVKDYFPEPVTVSWNSGALTSGVHTFPAV(L) (C)LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL(Y) (L)VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL(Y) (L)YSLSSVVTVPSSSLGTQT(Y) (L)YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEL(L) (L)YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSV(F) (L)YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVF(L) (L)YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL(M) (L)YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM(I) (T)YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSV(F) (L)LGGPSVFLFPPKPKDTLM(I) (V)FLFPPKPKDTLM(I) (F)LFPPKPKDTLM(I) (F)LFPPKPKDTLMISRTPEVTC(V) (L)MISRTPEVTC(V) (M)ISRTPEVTC(V) (M)ISRTPEVTCV(V) (C)VVVDVSHEDPEVKFNW(Y) (C)VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVL(T)* (C)VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVL(T)** 147 H28-93 H47-83 H57-83 H81-93 H84-104 H84-93 H84-95 H94-104 H95-104 H96-104 H105-115 H116-147 H116-148 H116-182 H148-176 H148-182 H149-182 H183-200 H183-237 H183-243 H183-244 H183-254 H183-255 H201-243 H238-255 H244-255 H245-255 H245-264 H255-264 H256-264 H256-265 H265-280 H265-309 H265-309 Table 3.4 (cont’d) 1799.8599 6606.0918 6703.5491 6907.606 7020.69 1229.5871 1128.5394 1025.5302 1437.7624 912.4462 1324.6783 3379.6481 2486.2198 3094.4276 2316.2135 4823.3821 4563.2296 2526.1864 2336.1452 (V)VVDVSHEDPEVKFNW(Y) (V)VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVL(T)*** (L)TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSL(T) (L)TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTC(L) (L)TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCL(V) (L)TCLVKGFYPSD(I) (T)CLVKGFYPSD(I) (C)LVKGFYPSD(I) (C)LVKGFYPSDIAVE(W) (L)VKGFYPSD(I) (L)VKGFYPSDIAVE(W) (L)VKGFYPSDIAVEWESNGQPENNYKTTPPVL(D) (D)IAVEWESNGQPENNYKTTPPVL(D) (D)IAVEWESNGQPENNYKTTPPVLDSDGSF(F) (F)FLYSKLTVDKSRWQQGNVF(S) (F)FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG(-) (L)YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG(-) (F)SCSVMHEALHNHYTQKSLSLSPG(-) (C)SVMHEALHNHYTQKSLSLSPG(-) H266-280 H266-309 H310-368 H310-370 H310-371 H369-379 H370-379 H371-379 H371-383 H372-379 H372-383 H372-401 H380-401 H380-407 H408-426 H408-449 H410-449 H427-449 H429-449 *6705.1696 is the monoisotopic mass for H265-309 with G0F glycosylation. **6867.2114 is the monoisotopic mass for H265-309 with G1F glycosylation. ***6606.0918 is the monoisotopic mass for H266-309 with G0F glycosylation. Table 3.5. Light- and heavy-chain peptides identified from a spin-membrane (spun at 500 g) peptic digest of Avastin. m/z of [M+H]+ 5388.6879 5923.9521 7439.6223 Peptide Sequence (-)DIQMTQSPSSLSASVGDRVTITCSASQDISNYL NWYQQKPGKAPKVLIY(F) (-)DIQMTQSPSSLSASVGDRVTITCSASQDISNYL NWYQQKPGKAPKVLIYFTSSL(H) (-)DIQMTQSPSSLSASVGDRVTITCSASQDISNYL NWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD(F ) 148 Amino Acids L1-49 L1-54 L1-70 Table 3.5 (cont’d) 8771.2832 8918.3517 9090.4364 9253.4998 2069.9523 3013.4174 3185.5022 3726.8625 3244.6459 3391.7144 5048.5503 5363.7119 2722.4563 2869.5247 4526.3606 4841.5223 1822.9222 2138.0838 1675.8537 1991.0154 10830.2625 7145.4419 9173.4266 6830.2803 8858.265 2047.0025 (-)DIQMTQSPSSLSASVGDRVTITCSASQDISNYL NWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDF TLTISSLQPED(F) (-)DIQMTQSPSSLSASVGDRVTITCSASQDISNYL NWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDF TLTISSLQPEDF(A) (-)DIQMTQSPSSLSASVGDRVTITCSASQDISNYL NWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDF TLTISSLQPEDFAT(Y) (-)DIQMTQSPSSLSASVGDRVTITCSASQDISNYL NWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDF TLTISSLQPEDFATY(Y) (Y)FTSSLHSGVPSRFSGSGSGTD(F) (L)HSGVPSRFSGSGSGTDFTLTISSLQPEDF(A) (L)HSGVPSRFSGSGSGTDFTLTISSLQPEDFAT(Y) (F)ATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVF(I) (Y)YCQQYSTVPWTFGQGTKVEIKRTVAAPSV(F) (Y)YCQQYSTVPWTFGQGTKVEIKRTVAAPSVF(I) (Y)YCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASV(V) (Y)YCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCL(L) (Q)YSTVPWTFGQGTKVEIKRTVAAPSV(F) (Q)YSTVPWTFGQGTKVEIKRTVAAPSVF(I) (Q)YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKS GTASV(V) (Q)YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKS GTASVVCL(L) (V)FIFPPSDEQLKSGTASV(V) (V)FIFPPSDEQLKSGTASVVCL(L) (F)IFPPSDEQLKSGTASV(V) (F)IFPPSDEQLKSGTASVVCL(L) (F)IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC(-) (V)VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACE(V) (V)VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC(-) (L)LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACE(V) (L)LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC(-) (E)VTHQGLSSPVTKSFNRGEC(-) 149 L1-82 L1-83 L1-85 L1-86 L50-70 L55-83 L55-85 L84-116 L87-115 L87-116 L87-132 L87-135 L91-115 L91-116 L91-132 L91-135 L116-132 L116-135 L117-132 L117-135 L117-214 L133-195 L133-214 L136-195 L136-214 L196-214 Table 3.5 (cont’d) 3562.6941 2097.1029 3165.5429 3312.6113 5573.7739 6630.2939 3495.6889 2427.2489 2280.1805 4147.8708 1075.5378 3091.3508 3065.5823 6165.1529 6637.3811 3118.5884 3590.8166 6425.1924 6572.2608 7839.9607 8826.4462 6046.056 1944.0663 1433.7861 1286.7177 2273.2032 1005.5034 998.4789 1898.9283 6705.1942 6867.244 (-)EVQLVESGGGLVQPGGSLRLSCAASGYTFTNY GMN(W) (N)WVRQAPGKGLEWVGWINT(Y) (N)WVRQAPGKGLEWVGWINTYTGEPTYAAD(F) (N)WVRQAPGKGLEWVGWINTYTGEPTYAADF(K) (N)WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF SLDTSKSTAYLQM(N) (N)WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF SLDTSKSTAYLQMNSLRAEDTAV(Y) (T)YTGEPTYAADFKRRFTFSLDTSKSTAYLQM(N) (D)FKRRFTFSLDTSKSTAYLQM(N) (F)KRRFTFSLDTSKSTAYLQM(N) (M)NSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGT L(V) (M)NSLRAEDTAV(Y) (V)YYCAKYPHYYGSSHWYFDVWGQGTL(V) (L)VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL(V) (L)VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVL(Q) (L)VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGL(Y) (L)VKDYFPEPVTVSWNSGALTSGVHTFPAVL(Q) (L)VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL(Y) (L)YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSV(F) (L)YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVF(L) (L)YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM(I) (L)YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTC(V) (T)YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLM(I) (L)LGGPSVFLFPPKPKDTLM(I) (V)FLFPPKPKDTLM(I) (F)LFPPKPKDTLM(I) (F)LFPPKPKDTLMISRTPEVTC(V) (M)ISRTPEVTC(V) (C)VVVDVSHED(P) (C)VVVDVSHEDPEVKFNW(Y) (C)VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVL(T)* (C)VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVL(T)** 150 H1-35 H36-53 H36-63 H36-64 H36-83 H36-93 H54-83 H64-83 H65-83 H54-118 H84-93 H94-118 H119-151 H119-180 H119-185 H152-180 H152-185 H186-246 H186-247 H186-258 H186-267 H204-258 H241-258 H247-258 H248-258 H248-267 H259-267 H268-276 H268-283 H268-312 H268-312 Table 3.5 (cont’d) 5725.726 6703.5491 6907.606 7020.69 7914.1183 1229.5871 1025.5302 4100.94 912.4462 3987.8559 2486.2198 3094.4276 2316.2135 4823.3821 4563.2296 4071.9552 2526.1864 (D)PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VL(T)*** (L)TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSL(T) (L)TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTC(L) (L)TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCL(V) (L)TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD(I) (L)TCLVKGFYPSD(I) (C)LVKGFYPSD(I) (C)LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS F(F) (L)VKGFYPSD(I) (L)VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF( F) (D)IAVEWESNGQPENNYKTTPPVL(D) (D)IAVEWESNGQPENNYKTTPPVLDSDGSF(F) (F)FLYSKLTVDKSRWQQGNVF(S) (F)FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG(K) (L)YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG(K) (L)TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G(K) (F)SCSVMHEALHNHYTQKSLSLSPG(K) H277-312 H313-371 H313-373 H313-374 H313-382 H372-382 H374-382 H374-410 H375-382 H375-410 H383-404 H383-410 H411-429 H411-452 H413-452 H417-452 H430-452 *6705.1942 is the monoisotopic mass for H268-312 with G0F glycosylation. **6867.244 is the monoisotopic mass for H268-312 with G1F glycosylation. ***5725.726 is the monoisotopic mass for H277-312 with G0F glycosylation. Table 3.6. Light- and heavy-chain peptides identified from a spin-membrane (spun at 500 g) peptic digest of Rituxan. m/z of [M+H]+ 5763.9302 4614.2544 7654.7282 3626.7849 2585.33 Peptide Sequence (-)QIVLSQSPAILSASPGEKVTMTCRASSSVSYIH WFQQKPGSSPKPWIYATSNL(A) (L)SASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWI YATSNL(A) (L)SASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWI YATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAAT(Y) (M)TCRASSSVSYIHWFQQKPGSSPKPWIYATSNL(A) (Y)IHWFQQKPGSSPKPWIYATSNL(A) 151 Amino Acids L1-53 L12-53 L12-84 L22-53 L32-53 Table 3.6 (cont’d) 3059.4916 3222.5549 1161.5746 2147.9855 3446.7202 5103.5561 5418.7177 3283.6568 5255.6544 2614.3988 2761.4672 4733.4647 1170.6841 1317.7525 2974.5884 3289.7501 1822.9222 2138.0838 1675.8537 1991.0154 9173.4266 6830.2803 8858.265 2047.0025 5393.702 7647.8403 8659.2912 9031.4743 (L)ASGVPVRFSGSGSGTSYSLTISRVEAEDAA T(Y) (L)ASGVPVRFSGSGSGTSYSLTISRVEAEDAAT Y(Y) (T)ISRVEAEDAAT(Y) (T)YYCQQWTSNPPTFGGGTKL(E) (T)YYCQQWTSNPPTFGGGTKLEIKRTVAAPSVF(I) (T)YYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSD EQLKSGTASV(V) (T)YYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSD EQLKSGTASVVCL(L) (Y)YCQQWTSNPPTFGGGTKLEIKRTVAAPSVF(I) (Y)YCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDE QLKSGTASVVCL(L) (Q)WTSNPPTFGGGTKLEIKRTVAAPSV(F) (Q)WTSNPPTFGGGTKLEIKRTVAAPSVF(I) (Q)WTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASVVCL(L) (L)EIKRTVAAPSV(F) (L)EIKRTVAAPSVF(I) (L)EIKRTVAAPSVFIFPPSDEQLKSGTASV(V) (L)EIKRTVAAPSVFIFPPSDEQLKSGTASVVCL(L) (V)FIFPPSDEQLKSGTASV(V) (V)FIFPPSDEQLKSGTASVVCL(L) (F)IFPPSDEQLKSGTASV(V) (F)IFPPSDEQLKSGTASVVCL(L) (V)VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC(-) (L)LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACE(V) (L)LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC(-) (E)VTHQGLSSPVTKSFNRGEC(-) (-)QVQLQQPGAELVKPGASVKMSCKASGYTFTSY NMHWVKQTPGRGLEWIG(A) (-)QVQLQQPGAELVKPGASVKMSCKASGYTFTSY NMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKA TL(T) (-)QVQLQQPGAELVKPGASVKMSCKASGYTFTSY NMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKA TLTADKSSSTAY(M) (-)QVQLQQPGAELVKPGASVKMSCKASGYTFTSY NMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKA TLTADKSSSTAYMQL(S) 152 L54-84 L54-85 L74-84 L85-103 L85-115 L85-131 L85-134 L86-115 L86-134 L90-114 L90-115 L90-134 L104-114 L104-115 L104-131 L104-134 L115-131 L115-134 L116-131 L116-134 L132-213 L135-194 L135-213 L195-213 H1-49 H1-70 H1-80 H1-83 Table 3.6 (cont’d) 9318.6224 10007.9092 1974.0187 4315.1613 6569.2996 7952.9336 8240.0817 5997.9327 3656.7901 995.4528 1418.5681 4332.1645 1194.6041 3118.5884 3590.8166 1943.0021 1812.9226 5886.8809 6397.1611 6544.2295 7811.9294 8798.4149 4092.9762 4750.3248 6018.0247 (-)QVQLQQPGAELVKPGASVKMSCKASGYTFTSY NMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKA TLTADKSSSTAYMQLSSL(T) (-)QVQLQQPGAELVKPGASVKMSCKASGYTFTSY NMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKA TLTADKSSSTAYMQLSSLTSEDSAV(Y) (E)LVKPGASVKMSCKASGYTF(T) (E)LVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRG LEWIG(A) (E)LVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRG LEWIGAIYPGNGDTSYNQKFKGKATL(T) (E)LVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRG LEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYM QL(S) (E)LVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRG LEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYM QLSSL(T) (F)TSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKF KGKATLTADKSSSTAYMQL(S) (G)AIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQL(S ) (L)SSLTSEDSAV(Y) (V)YYCARSTYYGGD(W) (D)WYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSG GTAALGCL(V) (L)VKDYFPEPVT(V) (L)VKDYFPEPVTVSWNSGALTSGVHTFPAVL(Q) (L)VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL(Y) (T)VSWNSGALTSGVHTFPAVL(Q) (L)YSLSSVVTVPSSSLGTQT(Y) (L)YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKA EPKSCDKTHTCPPCPAPEL(L) (L)YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKA EPKSCDKTHTCPPCPAPELLGGPSV(F) (L)YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKA EPKSCDKTHTCPPCPAPELLGGPSVF(L) (L)YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKA EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM(I) (L)YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKA EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTC(V) (T)YICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPE L(L) (T)YICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPE LLGGPSVF(L) (T)YICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLM(I) 153 H1-86 H1-93 H11-29 H11-49 H11-70 H11-83 H11-86 H30-83 H50-83 H84-93 H94-105 H106-149 H150-159 H150-178 H150-183 H160-178 H184-201 H184-238 H184-244 H184-245 H184-256 H184-265 H202-238 H202-245 H202-256 Table 3.6 (cont’d) 1944.0663 2930.5518 1433.7861 1286.7177 2273.2032 1005.5034 1104.5718 1898.9283 3721.8609 6705.1660 6867.2164 7029.2668 6671.577 6988.718 7882.1463 912.4462 1025.5302 3987.8559 2486.2198 3094.4276 2316.2135 4823.3821 4563.2296 4071.9552 2526.1864 (L)LGGPSVFLFPPKPKDTLM(I) (L)LGGPSVFLFPPKPKDTLMISRTPEVTC(V) (V)FLFPPKPKDTLM(I) (F)LFPPKPKDTLM(I) (F)LFPPKPKDTLMISRTPEVTC(V) (M)ISRTPEVTC(V) (M)ISRTPEVTCV(V) (C)VVVDVSHEDPEVKFNW(Y) (C)VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE(E) (C)VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVL(T)* (C)VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVL(T)** (C)VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVL(T)*** (L)TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSL(T) (L)TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCL(V) (L)TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD(I) (L)VKGFYPSD(I) (L)VKGFYPSDI(A) (L)VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF( F) (D)IAVEWESNGQPENNYKTTPPVL(D) (D)IAVEWESNGQPENNYKTTPPVLDSDGSF(F) (F)FLYSKLTVDKSRWQQGNVF(S) (F)FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG(K) (L)YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG(K) (L)TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G(K) (F)SCSVMHEALHNHYTQKSLSLSPG(K) *6705.1660 is the monoisotopic mass for H266-310 with G0F glycosylation. **6867.2164 is the monoisotopic mass for H266-310 with G1F glycosylation. ***7029.2668 is the monoisotopic mass for H266-310 with G2F glycosylation. 154 H239-256 H239-265 H245-256 H246-256 H246-265 H257-265 H257-266 H266-281 H266-297 H266-310 H266-310 H266-310 H311-369 H311-372 H311-380 H373-380 H373-381 H373-408 H381-402 H381-408 H409-427 H409-450 H411-450 H415-450 H428-450 Table 3.7. Light- and heavy-chain peptides identified from a spin-membrane (spun at 500 g) peptic digest of Vectibix. m/z of [M+H]+ 1206.5671 2524.2018 3239.5155 5054.4986 5830.8691 5943.9532 9116.4368 1336.6525 2051.9662 4643.3199 2549.3147 3325.6852 1834.001 2723.4555 3288.6986 3435.7671 5092.603 5407.7646 3125.6353 3272.7037 1822.9222 2138.0838 1675.8537 1991.0154 1877.9313 9173.4266 6830.2803 Peptide Sequence (-)DIQMTQSPSSL(S) (-)DIQMTQSPSSLSASVGDRVTITCQ(A) (-)DIQMTQSPSSLSASVGDRVTITCQASQDISN(Y) (-)DIQMTQSPSSLSASVGDRVTITCQASQDISNYL NWYQQKPGKAPKL(L) (-)DIQMTQSPSSLSASVGDRVTITCQASQDISNYL NWYQQKPGKAPKLLIYDASN(L) (-)DIQMTQSPSSLSASVGDRVTITCQASQDISNYL NWYQQKPGKAPKLLIYDASNL(E) (-)DIQMTQSPSSLSASVGDRVTITCQASQDISNYL NWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSG TDFTFTISSLQPEDIAT(Y) (L)SASVGDRVTITCQ(A) (L)SASVGDRVTITCQASQDISN(Y) (L)SASVGDRVTITCQASQDISNYLNWYQQKPGKAP KLLIYDASN(L) (Q)ASQDISNYLNWYQQKPGKAPKL(L) (Q)ASQDISNYLNWYQQKPGKAPKLLIYDASN(L) (N)YLNWYQQKPGKAPKL(L) (N)YLNWYQQKPGKAPKLLIYDASNL(E) (T)YFCQHFDHLPLAFGGGTKVEIKRTVAAPSV(F) (T)YFCQHFDHLPLAFGGGTKVEIKRTVAAPSVF(I) (T)YFCQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASV(V) (T)YFCQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCL(L) (Y)FCQHFDHLPLAFGGGTKVEIKRTVAAPSV(F) (Y)FCQHFDHLPLAFGGGTKVEIKRTVAAPSVF(I) (V)FIFPPSDEQLKSGTASV(V) (V)FIFPPSDEQLKSGTASVVCL(L) (F)IFPPSDEQLKSGTASV(V) (F)IFPPSDEQLKSGTASVVCL(L) (I)FPPSDEQLKSGTASVVCL(L) (V)VCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC(-) (L)LNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACE(V) 155 Amino Acids L1-11 L1-24 L1-31 L1-46 L1-53 L1-54 L1-85 L12-24 L12-31 L12-53 L25-46 L25-53 L32-46 L32-54 L86-115 L86-116 L86-132 L86-135 L87-115 L87-116 L116-132 L116-135 L117-132 L117-135 L118-135 L133-214 L136-195 Table 3.7 (cont’d) 8858.265 2047.0025 2093.1114 1641.8694 5485.6335 1240.642 1702.8898 1123.5677 2011.9477 2683.3243 3211.5973 1194.6041 3118.5884 3590.8166 2938.4945 1873.9178 6023.8745 6170.9429 7307.6023 7438.6427 4703.2183 5970.9182 4168.9745 4316.0429 5583.7428 3948.9227 (L)LNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC(-) (E)VTHQGLSSPVTKSFNRGEC(-) (-)QVQLQESGPGLVKPSETLSL(T) (L)QESGPGLVKPSETLSL(T) (L)TCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHI YYSGNTNYNPSLKSRL(T) (L)TISIDTSKTQF(S) (F)SLKLSSVTAADTAIYY(C) (Y)CVRDRVTGAF(D) (Y)CVRDRVTGAFDIWGQGTM(V) (M)VTVSSASTKGPSVFPLAPCSRSTSEST(A) (M)VTVSSASTKGPSVFPLAPCSRSTSESTAALGCL(V ) (L)VKDYFPEPVT(V) (L)VKDYFPEPVTVSWNSGALTSGVHTFPAVL(Q) (L)VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG L(Y) (F)PEPVTVSWNSGALTSGVHTFPAVLQSSGL(Y) (L)YSLSSVVTVPSSNFGTQT(Y) (L)YSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVD KTVERKCCVECPPCPAPPVAGPSV(F) (L)YSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVD KTVERKCCVECPPCPAPPVAGPSVF(L) (L)YSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVD KTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTL( M) (L)YSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVD KTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLM (I) (F)GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC PAPPVAGPSVF(L) (F)GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC PAPPVAGPSVFLFPPKPKDTLM(I) (T)YTCNVDHKPSNTKVDKTVERKCCVECPPCPAPP VAGPSV(F) (T)YTCNVDHKPSNTKVDKTVERKCCVECPPCPAPP VAGPSVF(L) (T)YTCNVDHKPSNTKVDKTVERKCCVECPPCPAPP VAGPSVFLFPPKPKDTLM(I) (C)NVDHKPSNTKVDKTVERKCCVECPPCPAPPVAG PSVF(L) 156 L136-214 L196-214 H1-20 H5-20 H21-69 H70-80 H81-96 H97-106 H97-114 H115-141 H115-147 H148-157 H148-176 H148-181 H153-181 H182-199 H182-238 H182-239 H182-249 H182-250 H196-239 H196-250 H200-238 H200-239 H200-250 H203-239 Table 3.7 (cont’d) 5216.6226 1433.7861 1286.7177 2273.2032 1173.6336 1136.5438 1005.5034 1598.7697 1898.8919 6019.7183 6181.7644 6673.1343 6835.1836 4139.8438 4301.8954 4793.2674 4955.3153 6705.5284 6909.5852 7022.6693 1229.5871 1025.5302 912.4462 2518.1919 3126.3997 2316.2135 2506.2547 4823.3821 2056.061 4563.2296 (C)NVDHKPSNTKVDKTVERKCCVECPPCPAPPVAG PSVFLFPPKPKDTLM(I) (V)FLFPPKPKDTLM(I) (F)LFPPKPKDTLM(I) (F)LFPPKPKDTLMISRTPEVTC(V) (L)FPPKPKDTLM(I) (L)MISRTPEVTC(V) (M)ISRTPEVTC(V) (C)VVVDVSHEDPEVQF(N) (C)VVVDVSHEDPEVQFNW(Y) (C)VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTF(R)* (C)VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTF(R)** (C)VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTFRVVSVL(T)*** (C)VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTFRVVSVL(T)**** (W)YVDGVEVHNAKTKPREEQFNSTF(R)***** (W)YVDGVEVHNAKTKPREEQFNSTF(R)****** (W)YVDGVEVHNAKTKPREEQFNSTFRVVSVL(T)** ***** (W)YVDGVEVHNAKTKPREEQFNSTFRVVSVL(T)** ****** (L)TVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKT KGQPREPQVYTLPPSREEMTKNQVSL(T) (L)TVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKT KGQPREPQVYTLPPSREEMTKNQVSLTC(L) (L)TVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKT KGQPREPQVYTLPPSREEMTKNQVSLTCL(V) (L)TCLVKGFYPSD(I) (C)LVKGFYPSD(I) (L)VKGFYPSD(I) (D)IAVEWESNGQPENNYKTTPPML(D) (D)IAVEWESNGQPENNYKTTPPMLDSDGSF(F) (F)FLYSKLTVDKSRWQQGNVF(S) (F)FLYSKLTVDKSRWQQGNVFSC(S) (F)FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG(K) (L)YSKLTVDKSRWQQGNVF(S) (L)YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG(K) 157 H203-250 H239-250 H240-250 H240-259 H241-250 H250-259 H251-259 H260-273 H260-275 H260-298 H260-298 H260-304 H260-304 H276-298 H276-298 H276-304 H276-304 H305-363 H305-365 H305-366 H364-374 H366-374 H367-374 H375-396 H375-402 H403-421 H403-423 H403-444 H405-421 H405-444 Table 3.7 (cont’d) 4071.9552 2526.1864 2336.1452 (L)TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG(K) (F)SCSVMHEALHNHYTQKSLSLSPG(K) (C)SVMHEALHNHYTQKSLSLSPG(K) H409-444 H422-444 H424-444 *6019.7183 is the monoisotopic mass for H260-298 with G0F glycosylation. **6181.7644 is the monoisotopic mass for H260-298 with G1F glycosylation. ***6673.1343 is the monoisotopic mass for H260-304 with G0F glycosylation. ****6835.1836 is the monoisotopic mass for H260-304 with G1F glycosylation. *****4139.8438 is the monoisotopic mass for H276-298 with G0F glycosylation. ******4301.8954 is the monoisotopic mass for H276-298 with G1F glycosylation. *******4793.2674 is the monoisotopic mass for H276-304 with G0F glycosylation. ********4955.3153 is the monoisotopic mass for H276-304 with G1F glycosylation. To test the reproducibility of spin proteolysis, we digested Av three times using three different spin columns. For the twenty highest signals in the mass spectra, standard deviations of the signal intensities (relative to the most intense peak in the spectrum) from triplicate digestion are <6% (Figure 3.12). We did not see signals of intact protein, and gel electrophoresis further confirms complete digestion of the four antibodies (see Figure 3.13). 158 Figure 3.12. Mass spectra of 3 different spin-membrane digests of Avastin. Each digestion employed a different peptic spin column. For the twenty highest signals in the mass spectra, standard deviations of the signals (relative to the most intense peak in the spectrum) from triplicate digestion are <6%. 159 Figure 3.13. Gel electrophoresis (SDS-PAGE) analysis of antibodies before and after digestion in a peptic spin column. Lanes 1 and 10: protein standards; Lanes 2, 4, 6 and 8: 5 Âľg of Herceptin, Avastin, Rituxan and Vectibix, respectively; Lanes 3, 5, 7 and 9: 5 Îźg of Herceptin, Avastin, Rituxan and Vectibix peptic spin digests (spun at 500 g), respectively. 3.3.4 mAb spin digestion with trypsin-containing membranes We previously digested an antibody using a trypsin membrane connected to a pipet tip, even without protein alkylation.46 However, reforming and/or scrambling of the disulfide bonds might occur under basic conditions. In developing a general protein pretreatment, we decided to conduct protein alkylation. Desalting is also necessary because of the large amount of denaturation and alkylation agents. We initially tried to desalt the reduced antibody before trypsin spin digestion. However, when desalting at the protein level using a C4 ZipTip, we did not detect identifiable peptides after spin digestion, presumably because of a large sample loss. 160 Zhao et al. found that reduced antibodies tend to precipitate during elution with 50% ACN and 0.1% FA.54 Considering the instability of reduced antibodies, we performed the desalting step after tryptic spin digestion. We were concerned that the salt and chaotropic agents in the digestion mixture, especially 0.8 M urea, might overcome electrostatic interactions between PSS and trypsin in the digestion membrane. However, this protocol yielded detectable tryptic peptides that cover 100% of the He, Av and Ve sequences, and 84% of the Ri sequence. Figure 3.14 presents the original mass spectrum of Avastin tryptic digestion, and Figure 3.15-3.18 give the sequence map of trypsin digestion of four antibodies. One of the missing pieces from Ri is H68-125, which has a theoretical monoisotopic mass of 6247.8700. The m/z of this peptide, which contains two missed cleavage sites, might be outside the mass range we set (300-1800). Another missing piece is H293-305, which contains the glycosylation site. A low ionization efficiency of these glycosylated peptides might explain why they did not give detectable signals in the MS spectrum. Prior studies indicate that in-solution tryptic digestion generates peptides with average lengths of ~14 amino acids. In contrast, in spin digestion, we found tryptic peptides with up to 10 missed cleavages. Based on 285 tryptic peptides identified from He, Av, Ri and Ve, the average antibody tryptic peptide length is 40 after spin digestion. Limited proteolysis time apparently leads to incomplete peptide digestion, but the larger peptides may facilitate characterization of antibody complementarity determining regions (CDRs). For example, a large tryptic peptide, L1108 of Ve, covers all the light chain CDRs, which makes characterization of three CDRs possible with a single peptide. As with peptic digestion, spin-membrane tryptic digestion enables identification of PTMs such as glycosylation, N-terminal glutamate formation and C-terminal Lysine clipping. Tables 3.8-3.11 present the full list of identified tryptic peptides. 161 Figure 3.14. Part of the mass spectrum of a tryptic spin digest of Avastin. Labels show the amino acids on the Hc and Lc. (not all of the peptide signals are labelled). 162 Figure 3.14 (cont’d) 163 Figure 3.14 (cont’d) 164 Figure 3.15. Sequence map of the peptides identified from infusion ESI-Orbitrap analysis of a tryptic digest of Herceptin. Light green color “N” represents the glycosylation site. 165 Figure 3.16. Sequence map of the peptides identified from infusion ESI-Orbitrap analysis of a tryptic digest of Avastin. Light green color “N” represents the glycosylation site. Red color “K” represents the C-terminal clipping. 166 Figure 3.17. Sequence map of the peptides identified from infusion ESI-Orbitrap analysis of a tryptic digest of Rituxan. Light green color “N” represents the glycosylation site. Red color “K” represents the C-terminal clipping. 167 Figure 3.18. Sequence map of the peptides identified from infusion ESI-Orbitrap analysis of a tryptic digest of Vectibix. Light green color “N” represents the glycosylation site. Red color “K” represents the C-terminal clipping. 168 Table 3.8. Light- and heavy-chain peptides identified from a spin-membrane (spun at 500 g) tryptic digest of Herceptin. m/z of [M+H]+ 2609.2658 7165.6262 11959.9174 2287.183 4575.3783 9369.6695 1772.9581 2307.2132 4657.2079 4813.309 7336.7223 3881.0055 4209.1801 1946.027 3724.9043 4053.079 4594.3803 10923.3997 11773.7389 2126.0699 Peptide Sequence (-)DIQMTQSPSSLSASVGDRVTITC(Carbamido methyl)R(A) (-)DIQMTQSPSSLSASVGDRVTITC(Carbamido methyl)RASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSR(S) (-)DIQMTQSPSSLSASVGDRVTITC(Carbamido methyl)RASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC(Carbamidom ethyl)QQHYTTPPTFGQGTKVEIKR(T) (R)ASQDVNTAVAWYQQKPGKAPK(L) (R)ASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSR(S) (R)ASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYC(Carbamidomethyl) QQHYTTPPTFGQGTKVEIKR(T) (K)LLIYSASFLYSGVPSR(F) (K)LLIYSASFLYSGVPSRFSGSR(S) (R)SGTDFTLTISSLQPEDFATYYC(Carbamidomethyl)QQH YTTPPTFGQGTKVEIK(R) (R)SGTDFTLTISSLQPEDFATYYC(Carbamidomethyl)QQH YTTPPTFGQGTKVEIKR(T) (K)VEIKRTVAAPSVFIFPPSDEQLKSGTASVVC(Carbamid omethyl)LLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSK(D) (K)RTVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidometh yl)LLNNFYPR(E) (K)RTVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidometh yl)LLNNFYPREAK(V) (R)TVAAPSVFIFPPSDEQLK(S) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPR(E) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPREAK(V) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPREAKVQWK(V) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYAC(Carbamidomethyl)EVT HQGLSSPVTK(S) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYAC(Carbamidomethyl)EVT HQGLSSPVTKSFNRGEC(Carbamidomethyl)(-) (K)SGTASVVC(Carbamidomethyl)LLNNFYPREAK(V) 169 Amino Acids L1-24 L1-66 L1-108 L25-45 L25-66 L25-108 L46-61 L46-66 L67-107 L67-108 L104-169 L108-142 L108-145 L109-126 L109-142 L109-145 L109-149 L109-207 L109-214 L127-145 Table 3.8 (cont’d) 7739.6777 1502.7584 2747.3457 3597.6849 2141.0808 1875.9269 2726.2661 1882.0029 4101.0975 5393.7964 6459.3208 2238.1124 3530.8114 5472.7811 1089.5476 2377.2411 895.4632 3576.6734 3951.8898 1186.6467 1321.678 8939.5187 7636.8585 3334.6421 4151.0585 (K)VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK SFNRGEC(Carbamidomethyl)(-) (K)DSTYSLSSTLTLSK(A) (K)ADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK (S) (K)ADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK SFNRGEC(Carbamidomethyl)(-) (K)HKVYAC(Carbamidomethyl)EVTHQGLSSPVTK(S) (K)VYAC(Carbamidomethyl)EVTHQGLSSPVTK(S) (K)VYAC(Carbamidomethyl)EVTHQGLSSPVTKSFNRGEC( Carbamidomethyl)(-) (-)EVQLVESGGGLVQPGGSLR(L) (-)EVQLVESGGGLVQPGGSLRLSC(Carbamidomethyl)AASGFNIKDTYIHWVR(Q) (-)EVQLVESGGGLVQPGGSLRLSC(Carbamidomethyl)AASGFNIKDTYIHWVRQAPGKGLEWVAR(I) (-)EVQLVESGGGLVQPGGSLRLSC(Carbamidomethyl)AASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN GYTR(Y) (R)LSC(Carbamidomethyl)AASGFNIKDTYIHWVR (Q) (R)LSC(Carbamidomethyl)AASGFNIKDTYIHWVRQAPGK GLEWVAR(I) (R)LSC(Carbamidomethyl)AASGFNIKDTYIHWVRQAPGK GLEWVARIYPTNGYTRYADSVKGR(F) (K)DTYIHWVR(Q) (R)QAPGKGLEWVARIYPTNGYTR(Y) (R)YADSVKGR(F) (R)FTISADTSKNTAYLQMNSLRAEDTAVYYC(Carbamido methyl)SR(W) (R)WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAP SSK(S) (K)GPSVFPLAPSSK(S) (K)STSGGTAALGC(Carbamidomethyl)LVK(D) (K)STSGGTAALGC(Carbamidomethyl)LVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYIC(Carbamidomethyl)NVNHKPSNTKVDKKVEPK(S) (K)DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYIC(Carbamidomethyl)NVNHKPSNTKV DKKVEPK(S) (K)SC(Carbamidomethyl)DKTHTC(Carbamidomethyl)PPC(C arbamidomethyl)PAPELLGGPSVFLFPPKPK(D) (K)SC(Carbamidomethyl)DKTHTC(Carbamidomethyl)PPC(C arbamidomethyl)PAPELLGGPSVFLFPPKPKDTLMISR(T) 170 L146-214 L170-183 L184-207 L184-214 L189-207 L191-207 L191-214 H1-19 H1-38 H1-50 H1-59 H20-38 H20-50 H20-67 H31-38 H39-59 H60-67 H68-98 H99-136 H125-136 H137-150 H137-221 H151-221 H222-251 H222-258 Table 3.8 (cont’d) 7929.8522 3797.8116 501.3144 2634.238 2516.333 1895.1324 838.5033 1267.762 1466.8941 4933.6642 4114.1787 3684.92 2343.1762 3485.7879 12088.8484 3047.554 1161.6296 2544.1314 4399.0354 5198.4906 7980.7398 8622.0783 6096.9647 2801.2671 3442.6056 (K)SC(Carbamidomethyl)DKTHTC(Carbamidomethyl)PPC(C arbamidomethyl)PAPELLGGPSVFLFPPKPKDTLMISRTPEV TC(Carbamidomethyl)VVVDVSHEDPEVKFNWYVDGVEV HNAK(T) (R)TPEVTC(Carbamidomethyl)VVVDVSHEDPEVKFNWYV DGVEVHNAK(T) (K)TKPR(E) (R)EEQYNSTYR(V)* (R)VVSVLTVLHQDWLNGKEYKC(Carbamidomethyl)K(V) (K)VSNKALPAPIEKTISKAK(G) (K)ALPAPIEK(T) (K)ALPAPIEKTISK(A) (K)ALPAPIEKTISKAK(G) (K)ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTC(Carbamidomethyl)LVK(G) (K)TISKAKGQPREPQVYTLPPSREEMTKNQVSLTC(Carba midomethyl)LVK(G) (K)AKGQPREPQVYTLPPSREEMTKNQVSLTC(Carbamido methyl)LVK(G) (K)GQPREPQVYTLPPSREEMTK(N) (K)GQPREPQVYTLPPSREEMTKNQVSLTC(Carbamidomet hyl)LVK(G) (K)GQPREPQVYTLPPSREEMTKNQVSLTC(Carbamidomet hyl)LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSC(Carbamidomethyl)SVMH EALHNHYTQKSLSLSPG(-) (R)EPQVYTLPPSREEMTKNQVSLTC(Carbamidomethyl)LV K(G) (K)NQVSLTC(Carbamidomethyl)LVK(G) (K)GFYPSDIAVEWESNGQPENNYK(T) (K)GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SK(L) (K)GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSR(W) (K)GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSC(Carbamidomethyl)SVMHEAL HNHYTQK(S) (K)GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSC(Carbamidomethyl)SVMHEAL HNHYTQKSLSLSPG(-) (K)TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC(Carb amidomethyl)SVMHEALHNHYTQKSLSLSPG(-) (R)WQQGNVFSC(Carbamidomethyl)SVMHEALHNHYTQK (S) (R)WQQGNVFSC(Carbamidomethyl)SVMHEALHNHYTQK SLSLSPG(-) *2634.238 is the monoisotopic mass for H296-304 with G0F glycosylation. 171 H222-291 H259-291 H292-295 H296-304 H305-325 H326-343 H330-337 H330-341 H330-343 H330-373 H338-373 H342-373 H344-363 H344-373 H344-449 H348-373 H364-373 H374-395 H374-412 H374-419 H374-442 H374-449 H396-449 H420-442 H420-449 Table 3.9. Light- and heavy-chain peptides identified from a spin-membrane (spun at 500 g) tryptic digest of Avastin. m/z of [M+H]+ 1878.8862 4957.4095 6701.3403 2801.3563 3097.5411 1762.9486 5285.5048 3881.0055 4209.1801 1946.027 3724.9043 4053.079 4594.3803 1797.8952 2126.0699 4489.1853 7217.5132 4161.0106 5032.4294 6889.3385 7739.6777 3619.7093 6348.0372 Peptide Sequence (-)DIQMTQSPSSLSASVGDR(V) (-)DIQMTQSPSSLSASVGDRVTITC(Carbamido methyl)SASQDISNYLNWYQQKPGKAPK(V) (-)DIQMTQSPSSLSASVGDRVTITC(Carbamido methyl)SASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSG VPSR(F) (R)VTITC(Carbamidomethyl)SASQDISNYLNWYQQKPGK( A) (R)VTITC(Carbamidomethyl)SASQDISNYLNWYQQKPGK APK(V) (K)VLIYFTSSLHSGVPSR(F) (R)FSGSGSGTDFTLTISSLQPEDFATYYC(Carbamidomethy l)QQYSTVPWTFGQGTKVEIKR(T) (K)RTVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidometh yl)LLNNFYPR(E) (K)RTVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidometh yl)LLNNFYPREAK(V) (R)TVAAPSVFIFPPSDEQLK(S) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPR(E) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPREAK(V) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPREAKVQWK(V) (K)SGTASVVC(Carbamidomethyl)LLNNFYPR(E) (K)SGTASVVC(Carbamidomethyl)LLNNFYPREAK(V) (R)EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSK(A) (R)EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSP VTK(S) (K)VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SK(A) (K)VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHK(V) (K)VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK (S) (K)VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK SFNRGEC(Carbamidomethyl)(-) (K)VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK(A) (K)VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK(S) 172 Amino Acids L1-18 L1-45 L1-61 L19-42 L19-45 L46-61 L62-108 L108-142 L108-145 L109-126 L109-142 L109-145 L109-149 L127-142 L127-145 L143-183 L143-207 L146-183 L146-190 L146-207 L146-214 L150-183 L150-207 Table 3.9 (cont’d) 1502.7584 2747.3457 3597.6849 2141.0808 2991.42 1875.9269 2726.2661 1882.0029 2197.9794 5178.4189 5334.52 2999.4574 3155.5585 2674.2936 1201.6212 1045.5201 1283.6412 5762.7213 1186.6467 1321.678 8015.9746 8358.165 8486.2599 8939.5187 6713.3145 7055.5048 (K)DSTYSLSSTLTLSK(A) (K)ADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK (S) (K)ADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK SFNRGEC(Carbamidomethyl)(-) (K)HKVYAC(Carbamidomethyl)EVTHQGLSSPVTK(S) (K)HKVYAC(Carbamidomethyl)EVTHQGLSSPVTKSFNRG EC(Carbamidomethyl)(-) (K)VYAC(Carbamidomethyl)EVTHQGLSSPVTK(S) (K)VYAC(Carbamidomethyl)EVTHQGLSSPVTKSFNRGEC( Carbamidomethyl)(-) (-)EVQLVESGGGLVQPGGSLR(L) (R)LSC(Carbamidomethyl)AASGYTFTNYGMNWVR(Q) (R)LSC(Carbamidomethyl)AASGYTFTNYGMNWVRQAPG KGLEWVGWINTYTGEPTYAADFK(R) (R)LSC(Carbamidomethyl)AASGYTFTNYGMNWVRQAPG KGLEWVGWINTYTGEPTYAADFKR(R) (R)QAPGKGLEWVGWINTYTGEPTYAADFK(R) (R)QAPGKGLEWVGWINTYTGEPTYAADFKR(R) (K)GLEWVGWINTYTGEPTYAADFKR(R) (R)RFTFSLDTSK(S) (R)FTFSLDTSK(S) (K)STAYLQMNSLR(A) (R)AEDTAVYYC(Carbamidomethyl)AKYPHYYGSSHWYF DVWGQGTLVTVSSASTKGPSVFPLAPSSK(S) (K)GPSVFPLAPSSK(S) (K)STSGGTAALGC(Carbamidomethyl)LVK(D) (K)STSGGTAALGC(Carbamidomethyl)LVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYIC(Carbamidomethyl)NVNHKPSNTK(V) (K)STSGGTAALGC(Carbamidomethyl)LVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYIC(Carbamidomethyl)NVNHKPSNTKVDK(K) (K)STSGGTAALGC(Carbamidomethyl)LVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYIC(Carbamidomethyl)NVNHKPSNTKVDKK(V) (K)STSGGTAALGC(Carbamidomethyl)LVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYIC(Carbamidomethyl)NVNHKPSNTKVDKKVEPK(S) (K)DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYIC(Carbamidomethyl)NVNHKPSNTK( V) (K)DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYIC(Carbamidomethyl)NVNHKPSNTKV DK(K) 173 L170-183 L184-207 L184-214 L189-207 L189-214 L191-207 L191-214 H1-19 H20-38 H20-65 H20-66 H39-65 H39-66 H44-66 H67-76 H68-76 H77-87 H88-139 H128-139 H140-153 H140-216 H140-219 H140-210 H140-224 H154-216 H154-219 Table 3.9 (cont’d) 7636.8585 3334.6421 4151.0585 6271.0681 7929.8522 2844.4575 4614.2279 2139.0274 3797.8116 1677.802 5325.5188 5613.679 6042.0424 2228.2074 2516.333 735.3818 1696.0003 1895.1324 838.5033 1267.762 1466.8941 4933.6642 2971.567 4114.1787 2542.3082 3684.92 6210.0335 (K)DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYIC(Carbamidomethyl)NVNHKPSNTKV DKKVEPK(S) (K)SC(Carbamidomethyl)DKTHTC(Carbamidomethyl)PPC(C arbamidomethyl)PAPELLGGPSVFLFPPKPK(D) (K)SC(Carbamidomethyl)DKTHTC(Carbamidomethyl)PPC(C arbamidomethyl)PAPELLGGPSVFLFPPKPKDTLMISR(T) (K)SC(Carbamidomethyl)DKTHTC(Carbamidomethyl)PPC(C arbamidomethyl)PAPELLGGPSVFLFPPKPKDTLMISRTPEV TC(Carbamidomethyl)VVVDVSHEDPEVK(F) (K)SC(Carbamidomethyl)DKTHTC(Carbamidomethyl)PPC(C arbamidomethyl)PAPELLGGPSVFLFPPKPKDTLMISRTPEV TC(Carbamidomethyl)VVVDVSHEDPEVKFNWYVDGVEV HNAK(T) (K)THTC(Carbamidomethyl)PPC(Carbamidomethyl)PAPELL GGPSVFLFPPKPK(D) (K)DTLMISRTPEVTC(Carbamidomethyl)VVVDVSHEDPEV KFNWYVDGVEVHNAK(T) (R)TPEVTC(Carbamidomethyl)VVVDVSHEDPEVK(F) (R)TPEVTC(Carbamidomethyl)VVVDVSHEDPEVKFNWYV DGVEVHNAK(T) (K)FNWYVDGVEVHNAK(T) (K)TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK(C)* (K)TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC(Car bamidomethyl)K(V)** (K)TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC(Car bamidomethyl)KVSNK(A)*** (R)VVSVLTVLHQDWLNGKEYK(C) (R)VVSVLTVLHQDWLNGKEYKC(Carbamidomethyl)K(V) (K)C(Carbamidomethyl)KVSNK(A) (K)VSNKALPAPIEKTISK(A) (K)VSNKALPAPIEKTISKAK(G) (K)ALPAPIEK(T) (K)ALPAPIEKTISK(A) (K)ALPAPIEKTISKAK(G) (K)ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTC(Carbamidomethyl)LVK(G) (K)TISKAKGQPREPQVYTLPPSREEMTK(N) (K)TISKAKGQPREPQVYTLPPSREEMTKNQVSLTC(Carba midomethyl)LVK(G) (K)AKGQPREPQVYTLPPSREEMTK(N) (K)AKGQPREPQVYTLPPSREEMTKNQVSLTC(Carbamido methyl)LVK(G) (K)AKGQPREPQVYTLPPSREEMTKNQVSLTC(Carbamido methyl)LVKGFYPSDIAVEWESNGQPENNYK(T) 174 H154-224 H225-254 H225-261 H225-280 H225-294 H229-254 H255-294 H262-280 H262-294 H281-294 H295-326 H295-328 H295-332 H308-326 H308-328 H327-332 H329-344 H329-346 H333-340 H333-344 H333-346 H333-376 H341-366 H341-376 H346-366 H345-376 H345-398 Table 3.9 (cont’d) 8621.2596 2343.1762 3485.7879 6010.9015 8665.2607 12091.8591 3047.554 5572.6676 1161.6296 6097.9692 2544.1314 4399.0354 5198.4906 7980.7398 2673.377 5455.6263 3442.6043 (K)AKGQPREPQVYTLPPSREEMTKNQVSLTC(Carbamido methyl)LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDK(S) (K)GQPREPQVYTLPPSREEMTK(N) (K)GQPREPQVYTLPPSREEMTKNQVSLTC(Carbamidomet hyl)LVK(G) (K)GQPREPQVYTLPPSREEMTKNQVSLTC(Carbamidomet hyl)LVKGFYPSDIAVEWESNGQPENNYK(T) (K)GQPREPQVYTLPPSREEMTKNQVSLTC(Carbamidomet hyl)LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSR(W) (K)GQPREPQVYTLPPSREEMTKNQVSLTC(Carbamidomet hyl)LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSC(Carbamidomethyl)SVMH EALHNHYTQKSLSLSPG(-) (R)EPQVYTLPPSREEMTKNQVSLTC(Carbamidomethyl)LV K(G) (R)EPQVYTLPPSREEMTKNQVSLTC(Carbamidomethyl)LV KGFYPSDIAVEWESNGQPENNYK(T) (K)NQVSLTC(Carbamidomethyl)LVK(G) (K)NQVSLTC(Carbamidomethyl)LVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDK(S) (K)GFYPSDIAVEWESNGQPENNYK(T) (K)GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SK(L) (K)GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSR(W) (K)GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSC(Carbamidomethyl)SVMHEAL HNHYTQK(S) (K)TTPPVLDSDGSFFLYSKLTVDKSR(W) (K)TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC(Carb amidomethyl)SVMHEALHNHYTQK(S) (R)WQQGNVFSC(Carbamidomethyl)SVMHEALHNHYTQK SLSLSPG(-) *5325.5188 is the monoisotopic mass for H295-326 with G0F glycosylation. **5613.679 is the monoisotopic mass for H295-328 with G0F glycosylation. ***6042.0424 is the monoisotopic mass for H295-332 with G0F glycosylation. 175 H345-420 H347-366 H347-376 H347-398 H347-422 H347-452 H351-376 H351-398 H367-376 H367-420 H377-398 H377-415 H377-422 H377-445 H399-422 H399-445 H423-452 Table 3.10. Light- and heavy-chain peptides identified from a spin-membrane (spun at 500 g) tryptic digest of Rituxan. m/z of [M+H]+ 2556.316 11556.7107 3724.9043 4053.079 10923.3997 11773.7389 2126.0699 7217.5132 4161.0106 6889.3385 7739.6777 2747.3457 3597.6849 2141.0808 2991.42 1875.9269 2726.2661 1960.0862 2466.2843 Peptide Sequence (-)QIVLSQSPAILSASPGEKVTMTC(Carbamido methyl)R(A) (-)QIVLSQSPAILSASPGEKVTMTC(Carbamido methyl)RASSSVSYIHWFQQKPGSSPKPWIYATSNLASGV PVRFSGSGSGTSYSLTISRVEAEDAATYYC(Carbamidomet hyl)QQWTSNPPTFGGGTKLEIKR(T) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPR(E) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPREAK(V) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYAC(Carbamidomethyl)EVT HQGLSSPVTK(S) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYAC(Carbamidomethyl)EVT HQGLSSPVTKSFNRGEC(Carbamidomethyl)(-) (K)SGTASVVC(Carbamidomethyl)LLNNFYPREAK(V) (R)EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSP VTK(S) (K)VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SK(A) (K)VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK (S) (K)VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK SFNRGEC(Carbamidomethyl)(-) (K)ADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK (S) (K)ADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK SFNRGEC(Carbamidomethyl)(-) (K)HKVYAC(Carbamidomethyl)EVTHQGLSSPVTK(S) (K)HKVYAC(Carbamidomethyl)EVTHQGLSSPVTKSFNRG EC(Carbamidomethyl)(-) (K)VYAC(Carbamidomethyl)EVTHQGLSSPVTK(S) (K)VYAC(Carbamidomethyl)EVTHQGLSSPVTKSFNRGEC( Carbamidomethyl)(-) (-)QVQLQQPGAELVKPGASVK(M) (-)QVQLQQPGAELVKPGASVKMSC(Carbamido methyl)K(A) 176 Amino Acids L1-24 L1-107 L108-141 L108-144 L108-206 L108-213 L126-144 L142-206 L145-182 L145-206 L145-213 L183-206 L183-213 L188-206 L188-213 L190-206 L190-213 H1-19 H1-23 Table 3.10 (cont’d) 4778.364 6942.3865 7402.6663 1186.6467 8911.4874 934.4299 3334.6421 4151.0585 7929.8522 2139.0274 3797.8116 3764.0568 1984.126 1696.0003 1895.1324 1267.762 1466.8941 4082.2067 3652.9479 3453.8159 12056.8763 8622.0783 (-)QVQLQQPGAELVKPGASVKMSC(Carbamido methyl)KASGYTFTSYNMHWVKQTPGR(G) (-)QVQLQQPGAELVKPGASVKMSC(Carbamido methyl)KASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGN GDTSYNQK(F) (-)QVQLQQPGAELVKPGASVKMSC(Carbamido methyl)KASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGN GDTSYNQKFKGK(A) (K)GPSVFPLAPSSK(S) (K)STSGGTAALGC(Carbamidomethyl)LVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYIC(Carbamidomethyl)NVNHKPSNTKVDKKAEPK(S) (K)AEPKSC(Carbamidomethyl)DK(T) (K)SC(Carbamidomethyl)DKTHTC(Carbamidomethyl)PPC(C arbamidomethyl)PAPELLGGPSVFLFPPKPK(D) (K)SC(Carbamidomethyl)DKTHTC(Carbamidomethyl)PPC(C arbamidomethyl)PAPELLGGPSVFLFPPKPKDTLMISR(T) (K)SC(Carbamidomethyl)DKTHTC(Carbamidomethyl)PPC(C arbamidomethyl)PAPELLGGPSVFLFPPKPKDTLMISRTPEV TC(Carbamidomethyl)VVVDVSHEDPEVKFNWYVDGVEV HNAK(T) (R)TPEVTC(Carbamidomethyl)VVVDVSHEDPEVK(F) (R)TPEVTC(Carbamidomethyl)VVVDVSHEDPEVKFNWYV DGVEVHNAK(T) (R)VVSVLTVLHQDWLNGKEYKC(Carbamidomethyl)KVS NKALPAPIEK(T) (K)C(Carbamidomethyl)KVSNKALPAPIEKTISK(A) (K)VSNKALPAPIEKTISK(A) (K)VSNKALPAPIEKTISKAK(G) (K)ALPAPIEKTISK(A) (K)ALPAPIEKTISKAK(G) (K)TISKAKGQPREPQVYTLPPSRDELTKNQVSLTC(Carba midomethyl)LVK(G) (K)AKGQPREPQVYTLPPSRDELTKNQVSLTC(Carbamido methyl)LVK(G) (K)GQPREPQVYTLPPSRDELTKNQVSLTC(Carbamidomet hyl)LVK(G) (K)GQPREPQVYTLPPSRDELTKNQVSLTC(Carbamidomet hyl)LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSC(Carbamidomethyl)SVMH EALHNHYTQKSLSLSPG(-) (K)GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSC(Carbamidomethyl)SVMHEAL HNHYTQKSLSLSPG(-) 177 H1-43 H1-63 H1-67 H126-137 H138-222 H219-226 H223-252 H223-259 H223-292 H260-278 H260-292 H306-338 H325-342 H327-342 H327-344 H331-342 H331-344 H339-374 H343-374 H345-374 H345-450 H375-450 Table 3.11. Light- and heavy-chain peptides identified from a spin-membrane (spun at 500 g) tryptic digest of Vectibix. m/z of [M+H]+ 4998.436 6727.3407 11874.817 3138.5677 1747.9225 6895.3989 5166.4942 2102.1281 3881.0055 4209.1801 1946.027 3724.9043 4053.079 4594.3803 10923.3997 11773.7389 2126.0699 8996.3906 7217.5132 Peptide Sequence (-)DIQMTQSPSSLSASVGDRVTITC(Carbamido methyl)QASQDISNYLNWYQQKPGKAPK(L) (-)DIQMTQSPSSLSASVGDRVTITC(Carbamido methyl)QASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSR(F) (-)DIQMTQSPSSLSASVGDRVTITC(Carbamido methyl)QASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISSLQPEDIATYFC(Carbamidome thyl)QHFDHLPLAFGGGTKVEIKR(T) (R)VTITC(Carbamidomethyl)QASQDISNYLNWYQQKPGK APK(L) (K)LLIYDASNLETGVPSR(F) (K)LLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIA TYFC(Carbamidomethyl)QHFDHLPLAFGGGTKVEIKR(T) (R)FSGSGSGTDFTFTISSLQPEDIATYFC(Carbamidomethyl) QHFDHLPLAFGGGTKVEIKR(T) (K)RTVAAPSVFIFPPSDEQLK(S) (K)RTVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidometh yl)LLNNFYPR(E) (K)RTVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidometh yl)LLNNFYPREAK(V) (R)TVAAPSVFIFPPSDEQLK(S) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPR(E) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPREAK(V) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPREAKVQWK(V) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYAC(Carbamidomethyl)EVT HQGLSSPVTK(S) (R)TVAAPSVFIFPPSDEQLKSGTASVVC(Carbamidomethyl )LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYAC(Carbamidomethyl)EVT HQGLSSPVTKSFNRGEC(Carbamidomethyl)(-) (K)SGTASVVC(Carbamidomethyl)LLNNFYPREAK(V) (K)SGTASVVC(Carbamidomethyl)LLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYAC(Carbamidomethyl)EVTHQGLSSPVTK(S) (R)EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSP VTK(S) 178 Amino Acids L1-45 L1-61 L1-108 L19-45 L46-61 L46-108 L62-108 L108-126 L108-142 L108-145 L109-126 L109-142 L109-145 L109-149 L109-207 L109-214 L127-145 L127-207 L143-207 Table 3.11 (cont’d) 8067.8524 4161.0106 6889.3385 7739.6777 3619.7093 6348.0372 2747.3457 3597.6849 2141.0808 1875.9269 2726.2661 7260.539 14701.312 2514.2293 3782.8629 1287.6514 1423.7097 8167.9492 8510.1395 9123.4943 6763.2573 (R)EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSP VTKSFNRGEC(Carbamidomethyl)(-) (K)VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SK(A) (K)VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK (S) (K)VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK SFNRGEC(Carbamidomethyl)(-) (K)VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK(A) (K)VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK(S) (K)ADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK (S) (K)ADYEKHKVYAC(Carbamidomethyl)EVTHQGLSSPVTK SFNRGEC(Carbamidomethyl)(-) (K)HKVYAC(Carbamidomethyl)EVTHQGLSSPVTK(S) (K)VYAC(Carbamidomethyl)EVTHQGLSSPVTK(S) (K)VYAC(Carbamidomethyl)EVTHQGLSSPVTKSFNRGEC( Carbamidomethyl)(-) (-)QVQLQESGPGLVKPSETLSLTC(Carbamido methyl)TVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYS GNTNYNPSLK(S) (-)QVQLQESGPGLVKPSETLSLTC(Carbamido methyl)TVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYS GNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYY C(Carbamidomethyl)VRDRVTGAFDIWGQGTMVTVSSAST KGPSVFPLAPC(Carbamidomethyl)SR(S) (R)DRVTGAFDIWGQGTMVTVSSASTK(G) (R)DRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPC(Car bamidomethyl)SR(S) (K)GPSVFPLAPC(Carbamidomethyl)SR(S) (R)STSESTAALGC(Carbamidomethyl)LVK(D) (R)STSESTAALGC(Carbamidomethyl)LVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQT YTC(Carbamidomethyl)NVDHKPSNTK(V) (R)STSESTAALGC(Carbamidomethyl)LVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQT YTC(Carbamidomethyl)NVDHKPSNTKVDK(T) (R)STSESTAALGC(Carbamidomethyl)LVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQT YTC(Carbamidomethyl)NVDHKPSNTKVDKTVERK(C) (K)DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSNFGTQTYTC(Carbamidomethyl)NVDHKPSNTK( V) 179 L143-214 L146-183 L146-207 L146-214 L150-183 L150-207 L184-207 L184-214 L189-207 L191-207 L191-214 H1-66 H1-135 H100-123 H100-135 H124-135 H136-149 H136-212 H136-215 H136-220 H150-212 Table 3.11 (cont’d) 7105.4477 3036.4966 3852.913 7631.6704 2908.4017 3724.8181 7503.5754 4614.1916 3797.7752 5279.5233 5567.649 9301.4297 2214.1917 2502.3173 2930.5557 3736.0255 1540.8516 1252.726 1681.9847 1911.1273 1253.7464 1482.889 3807.0473 4949.6591 (K)DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSNFGTQTYTC(Carbamidomethyl)NVDHKPSNTK VDK(T) (R)KC(Carbamidomethyl)C(Carbamidomethyl)VEC(Carbamid omethyl)PPC(Carbamidomethyl)PAPPVAGPSVFLFPPKPK(D ) (R)KC(Carbamidomethyl)C(Carbamidomethyl)VEC(Carbamid omethyl)PPC(Carbamidomethyl)PAPPVAGPSVFLFPPKPKD TLMISR(T) (R)KC(Carbamidomethyl)C(Carbamidomethyl)VEC(Carbamid omethyl)PPC(Carbamidomethyl)PAPPVAGPSVFLFPPKPKD TLMISRTPEVTC(Carbamidomethyl)VVVDVSHEDPEVQFN WYVDGVEVHNAK(T) (K)C(Carbamidomethyl)C(Carbamidomethyl)VEC(Carbamido methyl)PPC(Carbamidomethyl)PAPPVAGPSVFLFPPKPK(D) (K)C(Carbamidomethyl)C(Carbamidomethyl)VEC(Carbamido methyl)PPC(Carbamidomethyl)PAPPVAGPSVFLFPPKPKDT LMISR(T) (K)C(Carbamidomethyl)C(Carbamidomethyl)VEC(Carbamido methyl)PPC(Carbamidomethyl)PAPPVAGPSVFLFPPKPKDT LMISRTPEVTC(Carbamidomethyl)VVVDVSHEDPEVQFN WYVDGVEVHNAK(T) (K)DTLMISRTPEVTC(Carbamidomethyl)VVVDVSHEDPEV QFNWYVDGVEVHNAK(T) (R)TPEVTC(Carbamidomethyl)VVVDVSHEDPEVQFNWYV DGVEVHNAK(T) (K)TKPREEQFNSTFRVVSVLTVVHQDWLNGKEYK(C)* (K)TKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC(Car bamidomethyl)K(V)** (R)EEQFNSTFRVVSVLTVVHQDWLNGKEYKC(Carbamid omethyl)KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSR EEMTK(N)*** (R)VVSVLTVVHQDWLNGKEYK(C) (R)VVSVLTVVHQDWLNGKEYKC(Carbamidomethyl)K(V) (R)VVSVLTVVHQDWLNGKEYKC(Carbamidomethyl)KVS NK(G) (R)VVSVLTVVHQDWLNGKEYKC(Carbamidomethyl)KVS NKGLPAPIEK(T) (K)C(Carbamidomethyl)KVSNKGLPAPIEK(T) (K)VSNKGLPAPIEK(T) (K)VSNKGLPAPIEKTISK(T) (K)VSNKGLPAPIEKTISKTK(G) (K)GLPAPIEKTISK(T) (K)GLPAPIEKTISKTK(G) (K)GLPAPIEKTISKTKGQPREPQVYTLPPSREEMTK(N) (K)GLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS LTC(Carbamidomethyl)LVK(G) 180 H150-215 H220-246 H220-253 H220-286 H221-246 H221-253 H221-286 H247-286 H254-286 H287-318 H287-320 H291-358 H300-318 H300-320 H300-324 H300-332 H319-332 H321-332 H321-336 H321-338 H325-336 H325-338 H325-358 H325-368 Table 3.11 (cont’d) 3001.5775 4144.1893 2572.3188 3714.9306 2343.1762 3485.7879 6010.9015 11479.482 12120.8204 1904.9422 3047.554 5572.6676 1161.6296 6129.9413 5230.4626 8012.7119 8654.0503 5487.5984 4242.0608 2801.2671 3442.6056 (K)TISKTKGQPREPQVYTLPPSREEMTK(N) (K)TISKTKGQPREPQVYTLPPSREEMTKNQVSLTC(Carba midomethyl)LVK(G) (K)TKGQPREPQVYTLPPSREEMTK(N) (K)TKGQPREPQVYTLPPSREEMTKNQVSLTC(Carbamido methyl)LVK(G) (K)GQPREPQVYTLPPSREEMTK(N) (K)GQPREPQVYTLPPSREEMTKNQVSLTC(Carbamidomet hyl)LVK(G) (K)GQPREPQVYTLPPSREEMTKNQVSLTC(Carbamidomet hyl)LVKGFYPSDIAVEWESNGQPENNYK(T) (K)GQPREPQVYTLPPSREEMTKNQVSLTC(Carbamidomet hyl)LVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS FFLYSKLTVDKSRWQQGNVFSC(Carbamidomethyl)SVMH EALHNHYTQK(S) (K)GQPREPQVYTLPPSREEMTKNQVSLTC(Carbamidomet hyl)LVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS FFLYSKLTVDKSRWQQGNVFSC(Carbamidomethyl)SVMH EALHNHYTQKSLSLSPG(-) (R)EPQVYTLPPSREEMTK(N) (R)EPQVYTLPPSREEMTKNQVSLTC(Carbamidomethyl)LV K(G) (R)EPQVYTLPPSREEMTKNQVSLTC(Carbamidomethyl)LV KGFYPSDIAVEWESNGQPENNYK(T) (K)NQVSLTC(Carbamidomethyl)LVK(G) (K)NQVSLTC(Carbamidomethyl)LVKGFYPSDIAVEWESN GQPENNYKTTPPMLDSDGSFFLYSKLTVDK(S) (K)GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLY SKLTVDKSR(W) (K)GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLY SKLTVDKSRWQQGNVFSC(Carbamidomethyl)SVMHEAL HNHYTQK(S) (K)GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLY SKLTVDKSRWQQGNVFSC(Carbamidomethyl)SVMHEAL HNHYTQKSLSLSPG(-) (K)TTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC(Carb amidomethyl)SVMHEALHNHYTQK(S) (K)LTVDKSRWQQGNVFSC(Carbamidomethyl)SVMHEAL HNHYTQKSLSLSPG(-) (R)WQQGNVFSC(Carbamidomethyl)SVMHEALHNHYTQK (S) (R)WQQGNVFSC(Carbamidomethyl)SVMHEALHNHYTQK SLSLSPG(-) *5279.5233 is the monoisotopic mass for H287-318 with G0F glycosylation. **5567.649 is the monoisotopic mass for H287-320 with G0F glycosylation. ***9301.4297 is the monoisotopic mass for H291-358 with G0F glycosylation. 181 H333-358 H333-368 H337-358 H337-368 H339-358 H339-368 H339-390 H339-437 H339-444 H343-358 H343-368 H343-390 H359-368 H359-412 H369-414 H369-437 H369-444 H391-437 H408-444 H415-437 H415-444 3.3.5 LC/MS-MS analyses Direct infusion nanoESI is a powerful method for peptide mapping because of its short sample analysis time (<3 min for data collection). Also, injection of all peptides into the mass spectrometer avoids the peptide losses that are inevitable in LC.55 However, peptides have different ionization efficiencies,56 and ion suppression may occur during infusion MS analysis.57 Moreover, with protein mixtures spin digestion may generate hundreds or thousands of peptides, making effective direct infusion analysis impossible. LC-MS/MS is widely used to analyze complex protein mixtures, and its well-developed bioinformatics software makes data analysis possible. As an initial test of whether spinmembrane digestion enables analysis of antibody sequences and modifications using LC-MS/MS, we analyzed the antibody tryptic spin digests. (Peptic digests are much more difficult to analyze due to the limited cleavage specificity.49) Because mAbs are typically expressed in Chinese hamster ovary cell lines,58 we identified proteolytic peptides through comparison to the hamster reference proteome from Uniprot, with the addition of the mAb sequences to the database. Using MaxQuant data analysis with this protein data base, we compared the sequence coverage and number of unique peptides identified after tryptic spin digestion either in a spin membrane or in solution. 182 Table 3.12. Antibody Sequence Coverages and Numbers of Unique Peptides Obtained From LC/MS-MS Analyses of Tryptic Spin and In-solution Digests.* Sequence Coverage Unique Peptides Sequence Coverage Unique Peptides Tryptic Spin Digestion Herceptin Avastin Light Chain 100% 100% Heavy Chain 81.3% 85.2% Light Chain 40 41 Heavy Chain 66 72 Tryptic In-solution Digestion Herceptin Avastin Light Chain 87.4% 93.5% Heavy Chain 72.4% 73% Light Chain 15 17 Heavy Chain 33 33 Rituxan 100% 75.1% 30 43 Vectibix 100% 98.4% 37 52 Rituxan 93.0% 75.3% 17 32 Vectibix 94.9% 79.7% 14 30 *Peptides were identified using MaxQuant Software with comparison to a Chinese hamster proteome modified with antibody sequences. As Table 3.12 shows, for all four antibodies tryptic spin digestion gives higher or essentially equal sequence coverage and more unique peptides than in-solution digestion. The missing sequences in the light chain after in-solution digestion likely result from undetectable small peptides with 3 or 4 amino acids. Heavy-chain sequence coverages are lower in LC-MS/MS analysis than in direct infusion analysis, primarily because we didn’t consider the glycosylation on the heavy chain in the MaxQuant search. Glycosylated peptides also show low ionization efficiencies.59 Enzymatic removal of the glycans prior to digestion will likely give heavy-chain sequence coverages near 100%. In comparing in-solution and spin digestion, the additional unique peptides from spin digestion may enhance protein identification in database searching with protein mixtures. Overall, spin digestion is a powerful method for fast protein digestion, and proteolytic peptides from spin digestion are suitable for downstream direct infusion or LCMS/MS analysis. 183 3.4 Conclusions This work used pepsin/trypsin spin membranes as microreactors for reproducible proteolysis prior to MS analysis. The high concentration of enzyme in the membrane pores allows spin digestion of 100 ÂľL of antibody solution within 1 min. Peptic spin digestion avoids protein alkylation because the acidic conditions prevent reforming of disulfide bonds, whereas tryptic spin digestion benefits from alkylation. Moreover, with peptic spin digestion we can control the proteolytic peptide size by varying the spin rate. Direct infusion MS of spin digests is fast and provides a whole picture of the peptic/tryptic digests of single proteins. Essentially 100% peptide coverage along with identification of PTMSs results from direct infusion MS analyses of peptic and tryptic spin digests of Herceptin, Avastin, Rituxan and Vectibix. MaxQuant analyses of LCMS/MS data reveal that tryptic spin digests of four mAbs give higher sequence coverage and more unique peptides than in-solution tryptic digestion of the same antibodies. In summary, the spin-digestion platform is rapid, simple, and user-friendly, and it affords control over peptide sizes for various types of subsequent MS analyses. 3.5 Acknowledgement We gratefully acknowledge the U.S. National Science Foundation (CHE-1506315) for funding this work. We thank Dr. Mohammad Muhsin Chisti from Michigan State University for providing Herceptin, Avastin, Rituximab and Vectibix. We also thank Dr. Liangliang Sun (Michigan State University), Dr. Matthew Champion (Mass Spectrometry and Proteomics Facility of the University of Notre Dame) and Dr. Todd Lydic (Molecular Metabolism and Disease Collaborative Mass Spectrometry Core of Michigan State University) for helping to analyze the samples. 184 REFERENCES 185 REFERENCES (1) Angel, T. E.; Aryal, U. K.; Hengel, S. M.; Baker, E. S.; Kelly, R. T.; Robinson, E. W.; Smith, R. D. Mass spectrometry-based proteomics: existing capabilities and future directions. Chem. Soc. Rev. 2012, 41 (10), 3912. (2) Yates, J. R.; Ruse, C. I.; Nakorchevsky, A. Proteomics by mass spectrometry: approaches, advances, and applications. Annu. Rev. Biomed. Eng. 2009, 11, 49. (3) Monzo, A.; Sperling, E.; Guttman, A. Proteolytic enzyme-immobilization techniques for MS-based protein analysis. Trac-Trend Anal. Chem. 2009, 28 (7), 854. (4) Capelo, J. L.; Carreira, R.; Diniz, M.; Fernandes, L.; Galesio, M.; Lodeiro, C.; Santos, H. M.; Vale, G. Overview on modern approaches to speed up protein identification workflows relying on enzymatic cleavage and mass spectrometry-based techniques. Anal. Chim. Acta 2009, 650 (2), 151. (5) Ma, J. F.; Zhang, L. H.; Liang, Z.; Shan, Y. C.; Zhang, Y. K. Immobilized enzyme reactors in proteomics. Trac-Trend Anal. Chem. 2011, 30 (5), 691. (6) Switzar, L.; Giera, M.; Niessen, W. M. Protein digestion: an overview of the available techniques and recent developments. J. Proteome. Res. 2013, 12 (3), 1067. (7) Regnier, F. E.; Kim, J. Accelerating trypsin digestion: the immobilized enzyme reactor. Bioanalysis 2014, 6 (19), 2685. (8) Yang, H.; Zubarev, R. A. Mass spectrometric analysis of asparagine deamidation and aspartate isomerization in polypeptides. Electrophoresis 2010, 31 (11), 1764. (9) Zang, L.; Carlage, T.; Murphy, D.; Frenkel, R.; Bryngelson, P.; Madsen, M.; Lyubarskaya, Y. Residual metals cause variability in methionine oxidation measurements in protein pharmaceuticals using LC-UV/MS peptide mapping. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2012, 895-896, 71. (10) Jones, L. M.; Zhang, H.; Vidavsky, I.; Gross, M. L. Online, high-pressure digestion system for protein characterization by hydrogen/deuterium exchange and mass spectrometry. Anal. Chem. 2010, 82 (4), 1171. (11) http://www.sigmaaldrich.com/catalog/product/sigma/tt0010?lang=en®ion=US; 2016. (12) https://www.promega.com/products/mass-spectrometry/proteases-andsurfactants/trypsin-for-protein-characterization/trypsin-reagents/immobilizedtrypsin/?activeTab=2; Vol. 2016. 186 Vol. (13) Ma, J.; Zhang, L.; Liang, Z.; Zhang, W.; Zhang, Y. Recent advances in immobilized enzymatic reactors and their applications in proteome analysis. Anal. Chim. Acta 2009, 632 (1), 1. (14) Spross, J.; Sinz, A. A capillary monolithic trypsin reactor for efficient protein digestion in online and offline coupling to ESI and MALDI mass spectrometry. Anal. Chem. 2010, 82 (4), 1434. (15) Krenkova, J.; Lacher, N. A.; Svec, F. Highly efficient enzyme reactors containing trypsin and endoproteinase LysC immobilized on porous polymer monolith coupled to MS suitable for analysis of antibodies. Anal. Chem. 2009, 81 (5), 2004. (16) Ma, J.; Liang, Z.; Qiao, X.; Deng, Q.; Tao, D.; Zhang, L.; Zhang, Y. Organic-inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity. Anal. Chem. 2008, 80 (8), 2949. (17) Nicoli, R.; Gaud, N.; Stella, C.; Rudaz, S.; Veuthey, J. L. Trypsin immobilization on three monolithic disks for on-line protein digestion. J. Pharm. Biomed. Anal. 2008, 48 (2), 398. (18) Schoenherr, R. M.; Ye, M.; Vannatta, M.; Dovichi, N. J. CE-microreactor-CE-MS/MS for protein analysis. Anal. Chem. 2007, 79 (6), 2230. (19) Ota, S.; Miyazaki, S.; Matsuoka, H.; Morisato, K.; Shintani, Y.; Nakanishi, K. Highthroughput protein digestion by trypsin-immobilized monolithic silica with pipette-tip formula. J Biochem. Biophys. Methods 2007, 70 (1), 57. (20) Besanger, T. R.; Hodgson, R. J.; Green, J. R.; Brennan, J. D. Immobilized enzyme reactor chromatography: optimization of protein retention and enzyme activity in monolithic silica stationary phases. Anal. Chim. Acta 2006, 564 (1), 106. (21) Feng, S.; Ye, M.; Jiang, X.; Jin, W.; Zou, H. Coupling the immobilized trypsin microreactor of monolithic capillary with muRPLC-MS/MS for shotgun proteome analysis. J. Proteome. Res. 2006, 5 (2), 422. (22) Temporini, C.; Perani, E.; Mancini, F.; Bartolini, M.; Calleri, E.; Lubda, D.; Felix, G.; Andrisano, V.; Massolini, G. Optimization of a trypsin-bioreactor coupled with highperformance liquid chromatography-electrospray ionization tandem mass spectrometry for quality control of biotechnological drugs. J. Chromatogr. A 2006, 1120 (1-2), 121. (23) Massolini, G.; Calleri, E. Immobilized trypsin systems coupled on-line to separation methods: Recent developments and analytical applications. J. Sep. Sci. 2005, 28 (1), 7. (24) Kato, M.; Inuzuka, K.; Sakai-Kato, K.; Toyo'oka, T. Monolithic bioreactor immobilizing trypsin for high-throughput analysis. Anal. Chem. 2005, 77 (6), 1813. 187 (25) Krenkova, J.; Bilkova, Z.; Foret, F. Characterization of a monolithic immobilized trypsin microreactor with on-line coupling to ESI-MS. J. Sep. Sci. 2005, 28 (14), 1675. (26) Calleri, E.; Temporini, C.; Perani, E.; De Palma, A.; Lubda, D.; Mellerio, G.; Sala, A.; Galliano, M.; Caccialanza, G.; Massolini, G. Trypsin-based monolithic bioreactor coupled on-line with LC/MS/MS system for protein digestion and variant identification in standard solutions and serum samples. J. Proteome. Res. 2005, 4 (2), 481. (27) Ruan, G.; Wu, Z.; Huang, Y.; Wei, M.; Su, R.; Du, F. An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions. Biochem. Biophys. Res. Commun. 2016, 473 (1), 54. (28) Zhang, Z.; Sun, L.; Zhu, G.; Cox, O. F.; Huber, P. W.; Dovichi, N. J. Nearly 1000 Protein Identifications from 50 ng of Xenopus laevis Zygote Homogenate Using Online Sample Preparation on a Strong Cation Exchange Monolith Based Microreactor Coupled with Capillary Zone Electrophoresis. Anal. Chem. 2016, 88 (1), 877. (29) Sun, L.; Zhu, G.; Dovichi, N. J. Integrated capillary zone electrophoresis-electrospray ionization tandem mass spectrometry system with an immobilized trypsin microreactor for online digestion and analysis of picogram amounts of RAW 264.7 cell lysate. Anal. Chem. 2013, 85 (8), 4187. (30) Long, Y.; Wood, T. D. Immobilized pepsin microreactor for rapid peptide mapping with nanoelectrospray ionization mass spectrometry. J. Am. Soc. Mass. Spectrom. 2015, 26 (1), 194. (31) Yamaguchi, H.; Miyazaki, M.; Honda, T.; Briones-Nagata, M. P.; Arima, K.; Maeda, H. Rapid and efficient proteolysis for proteomic analysis by protease-immobilized microreactor. Electrophoresis 2009, 30 (18), 3257. (32) Sun, L.; Zhu, G.; Yan, X.; Mou, S.; Dovichi, N. J. Uncovering immobilized trypsin digestion features from large-scale proteome data generated by high-resolution mass spectrometry. J. Chromatogr. A 2014, 1337, 40. (33) Jeng, J.; Lin, M. F.; Cheng, F. Y.; Yeh, C. S.; Shiea, J. Using high-concentration trypsinimmobilized magnetic nanoparticles for rapid in situ protein digestion at elevated temperature. Rapid Commun. Mass Spectrom. 2007, 21 (18), 3060. (34) Li, Y.; Yan, B.; Deng, C.; Yu, W.; Xu, X.; Yang, P.; Zhang, X. Efficient on-chip proteolysis system based on functionalized magnetic silica microspheres. Proteomics 2007, 7 (14), 2330. (35) Slysz, G. W.; Lewis, D. F.; Schriemer, D. C. Detection and identification of subnanogram levels of protein in a nanoLC-trypsin-MS system. J. Proteome. Res. 2006, 5 (8), 1959. 188 (36) Slysz, G. W.; Schriemer, D. C. Blending protein separation and peptide analysis through real-time proteolytic digestion. Anal. Chem. 2005, 77 (6), 1572. (37) Freije, J. R.; Mulder, P. P.; Werkman, W.; Rieux, L.; Niederlander, H. A.; Verpoorte, E.; Bischoff, R. Chemically modified, immobilized trypsin reactor with improved digestion efficiency. J. Proteome. Res. 2005, 4 (5), 1805. (38) Moore, S.; Hess, S.; Jorgenson, J. Characterization of an immobilized enzyme reactor for on-line protein digestion. J. Chromatogr. A 2016, 1476, 1. (39) Liu, Y.; Lu, H.; Zhong, W.; Song, P.; Kong, J.; Yang, P.; Girault, H. H.; Liu, B. Multilayer-assembled microchip for enzyme immobilization as reactor toward low-level protein identification. Anal. Chem. 2006, 78 (3), 801. (40) Liuni, P.; Rob, T.; Wilson, D. J. A microfluidic reactor for rapid, low-pressure proteolysis with on-chip electrospray ionization. Rapid Commun. Mass Spectrom. 2010, 24 (3), 315. (41) Xu, F.; Wang, W. H.; Tan, Y. J.; Bruening, M. L. Facile trypsin immobilization in polymeric membranes for rapid, efficient protein digestion. Anal. Chem. 2010, 82 (24), 10045. (42) Tan, Y. J.; Wang, W. H.; Zheng, Y.; Dong, J.; Stefano, G.; Brandizzi, F.; Garavito, R. M.; Reid, G. E.; Bruening, M. L. Limited proteolysis via millisecond digestions in proteasemodified membranes. Anal. Chem. 2012, 84 (19), 8357. (43) Pang, Y.; Wang, W. H.; Reid, G. E.; Hunt, D. F.; Bruening, M. L. Pepsin-Containing Membranes for Controlled Monoclonal Antibody Digestion Prior to Mass Spectrometry Analysis. Anal. Chem. 2015, 87 (21), 10942. (44) Cooper, J. W.; Chen, J.; Li, Y.; Lee, C. S. Membrane-based nanoscale proteolytic reactor enabling protein digestion, peptide separation, and protein identification using mass spectrometry. Anal. Chem. 2003, 75 (5), 1067. (45) Gao, J.; Xu, J.; Locascio, L. E.; Lee, C. S. Integrated microfluidic system enabling protein digestion, peptide separation, and protein identification. Anal. Chem. 2001, 73 (11), 2648. (46) Ning, W.; Bruening, M. L. Rapid Protein Digestion and Purification with Membranes Attached to Pipet Tips. Anal. Chem. 2015, 87 (24), 11984. (47) Alves, N. J.; Champion, M. M.; Stefanick, J. F.; Handlogten, M. W.; Moustakas, D. T.; Shi, Y.; Shaw, B. F.; Navari, R. M.; Kiziltepe, T.; Bilgicer, B. Selective photocrosslinking of functional ligands to antibodies via the conserved nucleotide binding site. Biomaterials 2013, 34 (22), 5700. 189 (48) Fontana, A.; Zambonin, M.; Polverino de Laureto, P.; De Filippis, V.; Clementi, A.; Scaramella, E. Probing the conformational state of apomyoglobin by limited proteolysis. J. Mol. Biol. 1997, 266 (2), 223. (49) Ahn, J.; Cao, M. J.; Yu, Y. Q.; Engen, J. R. Accessing the reproducibility and specificity of pepsin and other aspartic proteases. Biochim. Biophys. Acta 2013, 1834 (6), 1222. (50) Hamuro, Y.; Coales, S. J.; Molnar, K. S.; Tuske, S. J.; Morrow, J. A. Specificity of immobilized porcine pepsin in H/D exchange compatible conditions. Rapid Commun. Mass Spectrom. 2008, 22 (7), 1041. (51) Zhang, Y.; Fonslow, B. R.; Shan, B.; Baek, M. C.; Yates, J. R., 3rd. Protein analysis by shotgun/bottom-up proteomics. Chem. Rev. 2013, 113 (4), 2343. (52) Beck, A.; Wagner-Rousset, E.; Ayoub, D.; Van Dorsselaer, A.; Sanglier-Cianferani, S. Characterization of therapeutic antibodies and related products. Anal. Chem. 2013, 85 (2), 715. (53) Daugherty, A. L.; Mrsny, R. J. Formulation and delivery issues for monoclonal antibody therapeutics. Adv. Drug Deliv. Rev. 2006, 58 (5-6), 686. (54) Zhao, Y.; Sun, L.; Knierman, M. D.; Dovichi, N. J. Fast separation and analysis of reduced monoclonal antibodies with capillary zone electrophoresis coupled to mass spectrometry. Talanta 2016, 148, 529. (55) Chen, S. Rapid protein identification using direct infusion nanoelectrospray ionization mass spectrometry. Proteomics 2006, 6 (1), 16. (56) Mirzaei, H.; Regnier, F. Enhancing electrospray ionization efficiency of peptides by derivatization. Anal. Chem. 2006, 78 (12), 4175. (57) Annesley, T. M. Ion suppression in mass spectrometry. Clin. Chem. 2003, 49 (7), 1041. (58) Li, F.; Vijayasankaran, N.; Shen, A. Y.; Kiss, R.; Amanullah, A. Cell culture processes for monoclonal antibody production. MAbs 2010, 2 (5), 466. (59) Song, E.; Pyreddy, S.; Mechref, Y. Quantification of glycopeptides by multiple reaction monitoring liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2012, 26 (17), 1941. 190 Chapter 4 . Membrane-base proteolytic digestion for protein sequence comparison Chapters 2 and 3 introduced protease-containing membranes for rapid protein digestion in syringe-pump and spin-column formats. With these platforms, protein digestion and subsequent peptide mapping give important information about the protein sequence. This chapter briefly explores the use of in-membrane digestion to compare two antibodies with similar sequences. 4.1 Introduction Because of their high specificity and long circulation lifetime in the body, mAbs are the fastest growing class of pharmaceutical drugs.1-3 However, the high cost of therapeutic mAbs is triggering the development of less-expensive biosimilar antibodies. These biosimilar antibodies have the same primary sequences as their originators, but they are normally expressed in different cell clones.3 Importantly, different glycosylation and heterogeneities of the biosimilar antibodies may affect their efficacy and safety.4 Rapid comparison of the biosimilar protein and its originator is crucial for biosimilar antibody research and development. Additionally unintended sequence variants that appear during antibody expression, purification and storage may introduce side effects to the patient. Detailed characterization of these sequence variants is important for quality control. Characterization of small sequence variations is also important in antibody engineering.5 Point mutations or additions of one or several amino acids to the primary sequence are common steps in mAb engineering to enhance antibody stability and affinity or prevent antibody aggregation. Robust sequence validation methods will facilitate this process, and protein digestion followed 191 by MS analysis can easily identify differences between biosimilar and originator antibodies6 and also verify antibody sequences before and after engineering. A number of studies used peptide mapping to analyze sequence variation in antibodies. Chen et al. employed Lys-C and trypsin digests and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to compare trastuzumab with two biosimilar antibodies. They identified protein posttranslational modifications and mutations in the biosimilar.7 Hongwei Xie and coworkers developed a rapid method for comparing a candidate biosimilar to an originator mAb using LC-MS technologies.8 Trypsin in-solution digestion and LC-MS with dataindependent acquisition successfully located a two amino acid residue variance in the heavy chain sequence of the biosimilar. Yang et al. compared the HPLC-UV/MS/MS tryptic peptide map of antibodies from four clones and found two sequence variants.9 Fu et al. identified alanine to serine sequence variants in an IgG4 by LC-MS tryptic peptide mapping.10 Similarly, Yantao Li and coworkers characterized alanine to valine sequence variants in the Fc region of a nivolumab biosimilar by LC-MS/MS after IdeS and trypsin digestion.11 Glaser and coworkers used trypsin and Lys-C digestion to confirm formation of the hinge region disulfides after antibody engineering.12 Rose et al. conducted hydrogen-deuterium exchange mass spectrometry with online pepsin digestion to study the structure change after mutation of Y407 in the CH3 domain of a human IgG,13 and Rui Gong and coworkers used MS to confirm the engineered human antibody CH2 region.14 Hussack et al. engineered a single-domain antibody with an additional disulfide bond to increase the protease resistance and thermal stability.15 They performed peptide mapping with LC-MS analysis of cyanogen bromide and trypsin digests. These studies demonstrate the power of MS technology for antibody sequence comparison. However, most of the aforementioned research used in-solution protein digestion, which usually 192 requires extensive sample preparation and long incubation times. Considering that in-membrane protein digestion can occur in as little as 1 min, I developed a workflow that includes proteolysis in pepsin-containing membranes and direct-infusion MS for rapid comparison of antibody sequences. Relative to LC-MS, direct infusion MS uses less sample analysis time. Simple comparison of two direct infusion ESI mass spectra takes less than 30 mins. The differences between the signals in two infusion mass spectra readily reveal sequence variations. 4.2 Experimental 4.2.1 Materials Nylon membranes (LoProdyne LP, nominal pore size 1.2 Îźm, 110 Îźm thickness) were purchased from Pall Corporation. c13C6FR1_ZMapp and c13C6FR1_ZMapp +K antibodies were obtained in their original formulations (Tris-acetate buffer with ≤ 200 mM Arginine, pH 7.12) as a gift from Dr. Adrian Guthals of Mapp Biopharmaceutical. Pepsin from porcine gastric mucosa (lyophilized powder, 3200-4500 units/mg protein), ammonium bicarbonate ( ≥ 99%), iodoacetamide (IAM, ≥ 99%), dithiothreitol (DTT, ≥ 99.5%), polystyrene sulfonate (PSS, average molecular weight ~70,000), and formic acid (FA, ≥98%) were purchased from Sigma Aldrich. Sequencing grade modified trypsin was obtained from Promega. NaCl (ACS grade) and HCl (ACS grade) were purchased from CCI. Other chemicals include urea (≥98%, Invitrogen), tris(2-carboxyethyl) phosphine hydrochloride (TCEP-HCl, >98%, Fluka), acetic acid (HOAc, ACS, Macron Fine Chemicals), and methyl alcohol (anhydrous, MeOH, Macron Fine Chemicals). Solutions were prepared in deionized water (DI water, Milli-Q, 18.2 MΊ¡cm at 25 °C). Amicon ultra 0.5 mL centrifugal filters (MWCO 10 kDa) were employed to desalt 193 samples before pepsin in-membrane and in-solution digestion, and for buffer exchange before trypsin in-solution digestion. An Eppendorf centrifuge (5415D) was used to conduct inmembrane digestion. 4.2.2 Manufacture of Pepsin-containing Membrane. The procedure for membrane fabrication is the same as described in Chapter 2. 4.2.3 Digestion of c13C6FR1_ZMapp and c13C6FR1_ZMapp +K antibodies with pepsin- containing membrane c13C6FR1_ZMapp and c13C6FR1_ZMapp +K antibodies were each diluted in deionized water to give 1 mg/mL antibody stock solutions. Subsequently, 2 ÂľL of 0.1 M HOAc and 2 ÂľL of 0.1 M TCEP-HCl were added to 20 ÂľL of mAb stock solutions. The reactions were incubated at 75 °C for 15 min. Buffer exchange with 5% FA employed 3 cycles of centrifugation with Amicon ultra 0.5 mL centrifugal filters (MWCO 10 kDa). About 25 ÎźL of solution remained after each centrifugation, and 475 ÎźL of 5% FA was added prior to the following centrifugation. Residues were diluted to 200 ÂľL with 5% FA to make 0.1 mg/mL antibody pre-digestion solutions. Pepsin in-membrane digestion of two antibodies was conducted by passing 200 ÎźL of each pre-digestion solution through a pepsin-containing membrane at 130 mL/h (syringe pump). Samples were dried in a SpeedVac, and reconstituted in MS buffer (50% MeOH, 49% H2O and 1% HOAc) for direct infusion MS analysis. 194 4.2.4 In-solution peptic digestion of c13C6FR1_ZMapp and c13C6FR1_ZMapp +K antibodies One ÎźL of 1 mg/mL pepsin solution was added to 200 ÂľL of 0.1 mg/mL antibody pre-digestion solution (prepared with buffer exchange as mentioned in the last subsection). The mixtures were incubated at 37 °C for 16 h, and the solutions were dried in a SpeedVac and reconstituted in MS buffer for MS analysis. 4.2.5 In-solution tryptic digestion of c13C6FR1_ZMapp and c13C6FR1_ZMapp +K antibodies Twenty ÂľL of 1 mg/mL mAb stock solution was dried and redissolved in 7 ÎźL of 2 mM TCEPHCl solution in 0.1% HOAc containing 8 M urea. The mixtures were incubated at 50 °C for 10 min prior to addition of 7 ÂľL of 20 mM IAM in 2 M NH4HCO3 containing 8 M urea, and incubation in the dark for 30 min. Finally, 6 ÂľL of 30 mM DTT in 100 mM NH4HCO3 containing 8 M urea was added followed by incubation in the dark for 20 min to quench the IAM. After reduction and alkylation, the residual solutions underwent buffer exchange with 10 mM NH4HCO3 using 3 centrifugation cycles. About 25 ÎźL of solution remained after each centrifugation, and 475 ÎźL of 10 mM NH4HCO3 was added prior to the following centrifugation. Finally, residues were diluted to 200 ÂľL with 10 mM NH4HCO3 to make 0.1 mg/mL alkylated antibody pre-digestion solutions. Then, 5 ÂľL of 0.2 Îźg/ÎźL sequencing grade modified trypsin solution was added to 200 ÎźL of the alkylated antibody solution prior to incubation at 37 °C for 16 h. The reaction was quenched by adding 5 ÎźL of acetic acid. Samples were then dried with a SpeedVac before reconstitution and infusion MS analysis. 195 4.2.6 Mass Spectrometry and Data Analysis The in-membrane digests and in-solution digests were dried with a SpeedVac and reconstituted in MS buffer within 1 day. Forty ÎźL of each sample was loaded into a Whatman multichem 96well plate (Sigma−Aldrich) and sealed with Teflon Ultrathin Sealing Tape (Analytical Sales and Services, Prompton Plains, NJ). An Advion Triversa Nanomate nanoelectrospray ionization (nESI) source (Advion, Ithaca, NY) was used to introduce the sample into a high-resolution accurate mass Thermo Fisher Scientific LTQ Orbitrap Velos mass spectrometer (San Jose, CA) that was equipped with a dual pressure ion trap, HCD cell, and ETD. The spray voltage and gas pressure were set to 1.4 kV and 1.0 psi, respectively. The ion source interface had an inlet temperature of 200 °C with an S-Lens value of 57%. High-resolution mass spectra were acquired in positive ionization mode across the m/z range of 300−1800, using the FT analyzer operating at a mass resolving power of 100,000. Spectra were the average of 100 scans. Signals with >1% of the highest peak intensities and S/N>3 were analyzed. Peptide identification was performed manually using comparison to masses obtained with ProteinProspector (v 5.14.1, University of California, San Francisco, CA). Mass tolerance was set to 10 ppm. 4.3 Results and discussion c13C6FR1_ZMapp (Z) and c13C6FR1_ZMapp +K (ZK) antibodies have similar primary sequences. The only difference is ZK has an extra Lysine on the light chain. Figure 4.1 shows part of the antibody sequences of Z and ZK to illustrate the sequence difference. 196 Figure 4.1. Parts of the sequences of 13C6FR1_ZMapp (Z) and c13C6FR1_ZMapp +K (ZK) antibodies. Interestingly, the extra Lysine is next to an Arginine. In this case, trypsin digestion is not the best option for identifying the extra lysine because the theoretical tryptic digest will contain the short peptide LELK. Even with a missed cleavage, the resulting peptide LELKR will still be small. Considering that this peptide contains two basic residues, it can carry as many as 3 protons. The m/z of the peptide will thus fall into the low m/z region (<200 m/z), which contains a large amount of noise. 4.3.1 Digestion of c13C6FR1_ZMapp and c13C6FR1_ZMapp +K antibodies in a pepsin-containing membrane Chapters 2 and 3 showed that digestion using pepsin-containing membranes enables rapid mAb characterization.16 The workflow is straightforward (Figure 4.2). In this particular application, we added a desalting step because the high salt concentration in the formulation might suppress the ionization of peptides.17 197 Figure 4.2. Workflow for comparison of two antibodies using pepsin-containing membranes for proteolysis. Digestion employed flow through the membrane at 130 mL/h, which corresponds to a 3 msec residence time with the assumption of 50% membrane porosity. The short residence time generates large peptides with missed cleavages, and passage of 200 ÂľL of protein solution through the membrane requires < 30 sec. We did not conduct peptide mapping of the two antibodies because our goal is simply to find the differences. If a peptide signal appears in one mass spectrum, but not another, we mark it as a peptide that potentially contains the different sequence. Figure 4.3 shows an example. The +2 peptide with an m/z of 474.2694 appears in the spectrum of Z but not ZK, while a +2 peptide with an m/z of 538.3162 is present in the spectrum of ZK but not Z. 198 Figure 4.3. Comparison of part of the mass spectra of in-membrane peptic digests of Z (top) and ZK (bottom). The deconvoluted mass difference between these two signals is 128.0936, which is the mass of a Lysine residue. Comparison of the peptide masses with the original antibody sequences identifies the m/z of 474.2694 with the Z light-chain peptide 107-115 (L107-115), and the m/z of 538.3162 with ZK L107-116. MS gives information about the peptide mass, and tandem mass spectrometry (MS/MS) provides peptide sequence information. Figure 4.4 shows the MS/MS spectra of these two peptides. 199 Figure 4.4. Comparison of part of the MS/MS spectra of L107-115 from Z (top) and L107116 from ZK (bottom). These spectra clearly show that the extra Lysine is located at the N-terminus of L107-116 from ZK, because the y9 ion of L107-116 has an m/z value of 947.5286 and the [M+H]+ m/z value of L107-115 from Z is 947.5310. It is not surprising that the MS/MS spectra show more b ions than y ions because of the basic residues at the N-terminus. Similarly, I compared the mass spectra of Z and ZK across the whole mass range. Table 4.1 presents all the identified differences from peptic digestion of Z and ZK. 200 Table 4.1. MS signals that correspond to differences in that analysis of peptic in-membrane digestion of Z and ZK. m/z 474.2694 538.3162 701.6310 762.6613 791.4129 828.1799 868.7954 868.9460 973.8495 1057.1447 1061.2066 1089.9587 Charge state +2 +2 +4 +4 +4 +4 +3 +4 +3 +5 +3 +5 Peptide from Z             Peptide from ZK             Amino Acid L107-115 L107-116 L91-116 L107-135 L88-116 L87-116 L107-131 L86-116 L107-134 L87-135 L87-115 L86-135 Remarkably, all of the signal differences identified from Z and ZK digestion cover the modification site. For example, L107-135 from ZK and L107-134 from Z as well as L87-116 from ZK and L87-115 from Z are also pairs of peptides with a mass difference corresponding to a Lysine. These results give unambiguous identification of the modification site. 4.3.2 In-solution peptic digestion of c13C6FR1_ZMapp and c13C6FR1_ZMapp +K antibodies For comparison, we also conducted in-solution pepsin digestion of the Z and ZK antibodies. Most of the peptide signals from in-solution digestion fall in the range of 400-900 m/z, whereas in-membrane digestion gives some larger peptides to cover the 400-1500 m/z range. Figure 4.5 gives an overview comparison of in-solution and in-membrane digestion of Z. 201 Figure 4.5. Mass spectra of in-solution (top) and in-membrane (bottom) digests of Z. The maximum intensity is 1.02 x 10E6 in the top spectrum and 8.64 x 10E5 in the bottom spectrum. When comparing the mass spectra of in-solution peptic digests of Z and ZK, the only difference is the absence of m/z 474.2696 (+2) and the addition of m/z 538.3169 (+2) in the spectrum of ZK. We did not see any of the other differences found with in-membrane digestion. Both in-solution and in-membrane peptic digestion show the difference between the two antibodies, but inmembrane digestion yields several signals that give more confidence in the identification. Moreover, membranes require only 1 min for digestion. 202 4.3.3 In-solution tryptic digestion of c13C6FR1_ZMapp and c13C6FR1_ZMapp +K antibodies In-solution trypsin digestion is the standard method for bottom-up proteomics. After reduction and alkylation, samples normally incubate in trypsin solutions at 37 °C overnight. We conducted tryptic in-solution digestion and used the strategy described above to compare the mass spectra of Z and ZK but didn’t find any differences in signals from multiply charged peptides in the two spectra. However, when I used the theoretical mass of LELR to check the Z spectrum, I found one single charge state signal at m/z 530.3301. Similarly, I found the peak correspond to singly charged LELK in the ZK spectrum (m/z 502.3240, +1). Figure 4.6 shows the comparison of analyses of tryptic in-solution digestion of Z and ZK. The signal of LELKR did not appear, presumably because of complete tryptic cleavage of the K-R bond. The signals of single-charged peptides appear in a noisy region, and it is not easy to differentiate whether a signal is from a peptide or a noise. 203 Figure 4.6. Part of the MS spectra of tryptic in-solution digests of Z (top) and ZK (bottom). This region shows different singly charged peaks. 4.4. Conclusion Comparison of two antibody sequences is important in antibody characterization and quality control. In-membrane digestion is a powerful method for rapid identification or verification of a modification site. Digestion of 200 ÂľL of protein solution requires as little as 30 sec, which prevents introduction of possible PTMs during long sample incubation times. Because of different missed cleavages, rapid peptic in-membrane digestion yields multiple peptides that contain the modification site. Compared with peptic in-solution digestion, in-membrane digestion gives more evidence of sequence differences and thus provides unambiguous identification of the modification. Tryptic in-solution digestion is not appropriate in the particular case of differentiating the Z and ZK antibodies, because the singly charged peptide ion 204 is hard to identify. In conclusion, peptic in-membrane antibody digestion is a powerful method for identifying sequence modifications. Direct infusion MS with fast manual interpretation (less than 1 h) saves sample analysis time compared with LC-MS/MS methods. 4.5 Acknowledgement We gratefully acknowledge the US National Science Foundation (CHE-1152762 and CHE1506315) for funding this work. We thank Dr. Adrian Guthals of Mapp Biopharmaceutical for providing c13C6FR1_ZMapp and c13C6FR1_ZMapp +K antibodies. We also thank Dr. Todd Lydic from the Molecular Metabolism and Disease Collaborative Mass Spectrometry Core for helping analyze the samples. 205 REFERENCES 206 REFERENCES (1) Weiner, G. J. Building better monoclonal antibody-based therapeutics. Nat. Rev. Cancer 2015, 15 (6), 361. (2) Weiner, L. M.; Surana, R.; Wang, S. Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat. Rev. Immunol. 2010, 10 (5), 317. (3) Beck, A.; Sanglier-Cianferani, S.; Van Dorsselaer, A. Biosimilar, biobetter, and next generation antibody characterization by mass spectrometry. Anal. Chem. 2012, 84 (11), 4637. (4) Beck, A.; Debaene, F.; Diemer, H.; Wagner-Rousset, E.; Colas, O.; Van Dorsselaer, A.; Cianferani, S. Cutting-edge mass spectrometry characterization of originator, biosimilar and biobetter antibodies. J. Mass Spectrom. 2015, 50 (2), 285. (5) Carter, P. Improving the efficacy of antibody-based cancer therapies. Nat. Rev. Cancer 2001, 1 (2), 118. (6) Zhang, Y.; Fonslow, B. R.; Shan, B.; Baek, M. C.; Yates, J. R., 3rd. Protein analysis by shotgun/bottom-up proteomics. Chem. Rev. 2013, 113 (4), 2343. (7) Chen, S. L.; Wu, S. L.; Huang, L. J.; Huang, J. B.; Chen, S. H. A global comparability approach for biosimilar monoclonal antibodies using LC-tandem MS based proteomics. J. Pharm. Biomed. Anal. 2013, 80, 126. (8) Xie, H.; Chakraborty, A.; Ahn, J.; Yu, Y. Q.; Dakshinamoorthy, D. P.; Gilar, M.; Chen, W.; Skilton, S. J.; Mazzeo, J. R. Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies. MAbs 2010, 2 (4), 379. (9) Yang, Y.; Strahan, A.; Li, C.; Shen, A.; Liu, H.; Ouyang, J.; Katta, V.; Francissen, K.; Zhang, B. Detecting low level sequence variants in recombinant monoclonal antibodies. MAbs 2010, 2 (3), 285. (10) Fu, J.; Bongers, J.; Tao, L.; Huang, D.; Ludwig, R.; Huang, Y.; Qian, Y.; Basch, J.; Goldstein, J.; Krishnan, R.et al. Characterization and identification of alanine to serine sequence variants in an IgG4 monoclonal antibody produced in mammalian cell lines. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2012, 908, 1. (11) Li, Y.; Fu, T.; Liu, T.; Guo, H.; Guo, Q.; Xu, J.; Zhang, D.; Qian, W.; Dai, J.; Li, B.et al. Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells. MAbs 2016, 8 (5), 951. 207 (12) Glaser, S. M.; Hughes, I. E.; Hopp, J. R.; Hathaway, K.; Perret, D.; Reff, M. E. Novel antibody hinge regions for efficient production of CH2 domain-deleted antibodies. J. Biol. Chem. 2005, 280 (50), 41494. (13) Rose, R. J.; van Berkel, P. H.; van den Bremer, E. T.; Labrijn, A. F.; Vink, T.; Schuurman, J.; Heck, A. J.; Parren, P. W. Mutation of Y407 in the CH3 domain dramatically alters glycosylation and structure of human IgG. MAbs 2013, 5 (2), 219. (14) Gong, R.; Vu, B. K.; Feng, Y.; Prieto, D. A.; Dyba, M. A.; Walsh, J. D.; Prabakaran, P.; Veenstra, T. D.; Tarasov, S. G.; Ishima, R.et al. Engineered human antibody constant domains with increased stability. J. Biol. Chem. 2009, 284 (21), 14203. (15) Hussack, G.; Hirama, T.; Ding, W.; Mackenzie, R.; Tanha, J. Engineered single-domain antibodies with high protease resistance and thermal stability. PLoS One 2011, 6 (11), e28218. (16) Pang, Y.; Wang, W. H.; Reid, G. E.; Hunt, D. F.; Bruening, M. L. Pepsin-Containing Membranes for Controlled Monoclonal Antibody Digestion Prior to Mass Spectrometry Analysis. Anal. Chem. 2015, 87 (21), 10942. (17) Metwally, H.; McAllister, R. G.; Konermann, L. Exploring the mechanism of saltinduced signal suppression in protein electrospray mass spectrometry using experiments and molecular dynamics simulations. Anal. Chem. 2015, 87 (4), 2434. 208 Chapter 5 . Summary and future work 5.1 Research summary This dissertation describes the development of protease-containing membranes for rapid, controlled protein digestion prior to MS analysis. Most of my work focuses on monoclonal antibody digestion. I conducted in-membrane digestion using a prototype setup with a syringe pump, as well as with a novel spin membrane. In-membrane digestion removes the bottleneck of conventional in-solution digestion, which requires extensive time and labor, in workflows for “bottom-up” or ‘middle-down” protein analysis. Chapter 2 describes the use of pepsin-containing membrane as controlled reactors for monoclonal antibody digestion. Pepsin is an inexpensive protease that enables membrane digestion in acidic conditions, which avoids the need for antibody alkylation and minimizes protein modification during digestion. Layer-by-layer adsorption of PSS and pepsin in nylon membranes generates an IMER, and pepsin immobilization in membrane pores yields a high local enzyme concentration (~ 70 mg per mL of membrane) that enables digestion of 100 ÎźL of antibody solution in less than one minute. Moreover, in-membrane digestion using a high flow rate (130 mL/h) yields relatively large peptides (5-15 kDa) that cover the entire antibody sequence. As needed, digestion with different flow rates can enhance sequence coverage. Pepsin digestion followed by infusion MS analysis gives nearly 100% sequence coverage for both a WatersTM antibody and Herceptin. Additionally, MS analysis of the proteolytic peptides reveals sites for oxidation, deamidation and N-terminal pyroglutamic acid formation, as well as glycosylation patterns. Furthermore, CID, HCD and ETD MS/MS of the light-chain peptides generated from in-membrane digestion cleave 99% of the amino acid bonds in the light chain. 209 For comparison, “Top-down” analysis of the entire light chain by MS/MS methods shows a sequence coverage of only 55%. With minimal sample preparation time, membrane digestion leads to high peptide and sequence coverages for identification of PTMs by MS. Chapter 3 introduces a novel platform for in-membrane digestion. Membrane-based digestion methods developed by previous group members required a syringe pump and Upchurch fittings that may limit widespread adoption of the technique. I employed a spin membrane to greatly simplify the method, and these membranes will likely be commercially available in 2017. Protease-containing membranes inserted into spin columns enable digestion using simple centrifugation to pass the protein solutions through the membrane. Centrifuging 100-200 ÂľL of pretreated protein solution through a membrane requires 1 min or less. We tested the performance of spin digestion on apomyoglobin and four commercialized antibodies (Herceptin, Avastin, Rituxan and Vectibix). Direct infusion analysis of peptic and tryptic digests from these proteins gives nearly 100% sequence coverage. Protein PTMs, such as glycosylation, C-terminal Lysine clipping, and pyroglutamate formation, can be easily identified. Variation of the spin rates yields different proteolytic peptide sizes for apomyoglobin. Fast spin rates generate large peptides with more missed cleavages. LC-MS/MS analysis of antibody tryptic digests followed by MaxQuant data analysis reveals 100% sequence coverages for all the antibody light chains, and 75.1% to 98.4% coverage for the heavy chains. Compared with in-solution digestion, tryptic spin digestion gives higher sequence coverages and more unique peptides. Chapter 4 explores the application of a pepsin-containing membrane to comparison of two antibodies with similar sequences. One antibody has an extra Lysine. In-membrane peptic digestion and MS analysis of the two antibodies give multiple evidences for the sequence variation. Analysis of the MS signals of multiply charged peptides that appear exclusively in the 210 mass spectrum of only one of the antibodies shows that all of these peptides contain the sequence variation region. Tandem mass spectrometry analysis of these peptides further identifies the location of the variation. In contrast, in-solution peptic digestion generates only one peptide with the sequence difference, whereas in-solution trypsin digestion gives a four-amino acid peptide that shows a +1 charge state in the mass spectrum and is difficult to distinguish from the MS noise. Rapid in-membrane digestion (<30 sec) with direct infusion MS analysis is a time-saving workflow for protein sequence comparison. 5.2 Future work 5.2.1 Limited proteolysis in protease-containing membranes to interrogate protein higher order structure The structure of a protein essentially determines its function. Generally, proteins have four levels of structure: primary, secondary, tertiary and quaternary. The primary level is the linear amino acid sequence, and secondary structure refers to local conformations such as Îą-helices and βsheets. Tertiary structure is the three-dimensional shape of a protein molecule, whereas the quaternary level refers to the geometry of complexes that contain multiple protein subunits. Studies of the structures of proteins are crucial for understanding their function and interaction with other proteins, and limited digestion can facilitate the identification of protein conformational changes.1 In-membrane digestion is a promising method for studying protein higher order structures. For this aim, trypsin-containing membranes are preferable to pepsin-modified membranes, because peptic digestion occurs at acidic pH, which will partially denature the protein. Former group 211 member Dr. Yujing Tan conducted limited membrane-based proteolysis to locate the flexible region of Root Hair Defective 3 (RHD3) protein.2 In-membrane digestion of RHD3 with trypsin and chymotrypsin under nondenaturing conditions reveals a region around R672 that is highly flexible because peptide 673-691 (protein full length 1-691) shows the strongest signal in the mass spectrum of the protein after rapid in-membrane digestion. Chapter 4 provides an example of comparing protein primary sequences using proteasecontaining membranes and direct-infusion MS. Similarly, we could employ in-membrane digestion to study changes in protein higher order structure.3-5 Theoretically, digestion of the same protein with two different conformations will give different digestion patterns because protein conformation affects the accessibility to the proteolytic enzymes. To explore this possibility, we performed in-membrane digestion on apo and holo proteins with different conformations. Dr. Geiger’s group at Michigan State University recently studied the structure of cellular retinol binding proteins (CRBPs). They found that the crystal structure of human CRBPII changes after binding of retinal. However, data from X-ray diffraction represent the protein structure in the solid state. MS has been widely used to study protein structures in solution.6,7 Dr. Geiger provided us with two CRBPII proteins, one is the apo domain swapped (DS) dimer, and the other is the holo DS dimer, the apo DS dimer with a bound retinal. For this particular case, we conducted in-membrane digestion of the two proteins with a membrane containing trypsin immobilized through a covalent linkage. Trypsin is highly active, and covalent immobilization of this enzyme decreases its activity, which should facilitate limited trypsinolysis. We fabricate such IMERs by adsorption of poly (acrylic acid) (PAA) in membranes followed by activation of –COOH groups with NHS/EDC and covalent coupling via amide linkages. Direct infusion mass 212 spectra of tryptic digests (1.5-sec residence times) of the apo and holo proteins showed different digestion patterns. Figure 5.1 represents the original mass spectra. Figure 5.1. Direct infusion MS spectra of in-membrane tryptic digests (1.5-sec residence times) of Apo and Holo DS CRBPII dimers. In general, the apo DS dimer undergoes more proteolytic digestion than the holo DS dimer. Intact holo DS dimer signals dominate the mass spectrum of this protein, which suggests that the holo DS dimer has a rigid structure that resists trypsin proteolysis. Apo monomer is a protein with 133 amino acids. Tryptic peptides, 3-21, 22-30, 36-58, 59-80, 84-104, and 109-127, nearly cover the apo sequence. However, only three peptides, 22-30, 36-58 and 41-58, can be identified in the MS spectrum from the holo tryptic digest. These evidences are consistent with a conformational change in the apo DS dimer after binding retinal molecules. N59 is a key amino acid for this conformational change because amino acids after 59 are missing. Future work 213 should digest the two proteins with a membrane containing electrostatically immobilized trypsin. Electrostatic immobilization leads to higher enzyme activity than covalent binding and should provide more digestion on the holo DS dimers. Also, we can conduct the covalent/electrostatic trypsin digestion of the two proteins under different flow rates to potentially identify the most flexible regions of these dimers. 5.2.2 Polyclonal antibody digestion by protease-containing membranes Monoclonal antibodies and related products represent the largest portion of the biologic therapeutics market.8 Antibody engineering technology is widely used to enhance ligand binding affinity, reduce immunogenicity, and optimize in vivo half-life.9 However, these engineered products do not necessarily have improved clinical efficacy. Motavizumab, for example, is engineered from Palivizumab and shows 75 times greater affinity for respiratory syncytial virus F protein than its parent antibody. However, a phase-2, randomized, double-blind safety and pharmacokinetic assessment of these two antibodies showed similar results on high-risk children.10 To overcome such challenges, researchers began developing multi-specific antibodies (antibodies that can bind two distinct epitopes), oligoclonal cocktails (mixtures of two or three monoclonal antibodies), and recombinant polyclonal antibodies (a single master cell line express a single light chain and up to five heavy chains).11 An approved biclonal combination of Trastuzumab (Herceptin) and Pertuzumab (Perjeta), which target different epitopes on the HER2 growth factor receptor, synergistically inhibited the survival of BT474 cells.12 Similar to monoclonal antibodies, these polyclonal antibodies require extensive characterization prior to distribution to patients. 214 Characterization is more difficult for polyclonal antibodies than for mAbs. Top-down analysis will be challenging, and MS of antibody subunits might reveals antibody PTMs, but specifying the PTM sites is still challenging. Bottom-up analysis of polyclonal antibodies yields tryptic peptides from multiple antibodies, and it is hard to identify the origin of a single peptide because these antibodies share a large portion of similar sequences. Rapid in-membrane peptic digestion has the unique advantages of generating large peptides whose origin may be easier to determine. To investigate in-membrane digestion for analysis of polyclonal antibodies, I digested a mixture of four equimolar antibodies with a pepsin-containing membrane using a residence time of 3 msec. Figure 5.2 shows the mass spectrum of the digest. Figure 5.2. Mass spectra of an in-membrane peptic digest (3-msec residence time) of a cocktail of four antibodies. Multiple labels represent all the possible origins of an identified peptide. Not all identified peptide are labeled with sequences. The 3-mec peptic in-membrane digest gives a large amount of peptides, and most of the signals in Figure 5-2 stem from the four antibodies. Rapid peptic digestion yields large peptides, for 215 example, L87-215 from mAb2. Not surprisingly, most of the identified signals were from antibody light and heavy chain constant regions, however, because these four antibodies share a large percentage of the same sequence, especially in the constant region. Ion suppression may occur for the peptides from light- and heavy-chain variable regions. LC-MS/MS analysis of the digests might be a better choice, not only because manual data interpretation is very timeconsuming in this case, but also because separation of the peptides would solve the ionsuppression problem. Future work should focus on developing a straightforward data analysis workflow and investigating the LC-MS/MS analysis method. 5.2.3 De novo antibody sequencing Discovery and development of a therapeutic monoclonal antibody normally begins with screening for high affinity antibodies and subsequent sequencing and production of these targets. Identification of novel antibodies normally depends on DNA sequencing or hybridoma cloning of peripheral B cells.13 However, the antibody DNA does not necessarily predict the antibody primary sequence because of V(D)J recombination during B cell maturation, and somatic hypermutation during B cell affinity maturation results in extremely diverse sequences.14 Hybridoma cloning of peripheral B cells is a powerful method, but not all antibodies are produced by peripheral B cells. Long-lived plasma B cells, for instance, reside in bone marrow where sampling will be challenging. De novo antibody sequencing is necessary when DNA of the antibody or a B cell is not available. MS-based protein sequencing methods have developed greatly over the past 30 years. Biemann et al. first obtained the sequence of thioredoxin in1987 by sequencing 14 tryptic peptides and several peptides from Staphylococcus aureus protease.15 The Biemann group further employed 216 trypsin digestion and thermolysin digestion on glutaredoxin and deduced the sequence using CID mass spectra.16 Peptides generated from one protease overlap with peptides from other proteases, and thus, MS/MS of these peptides provides important information about the peptide arrangement. Nuno Bandeira et al. developed the shotgun protein sequencing (SPS) method to detect, score and interpret overlaps between uninterpreted MS/MS spectra.17 Bandeira et al. further developed a comparative shotgun protein sequencing method (CSPS).18 Alignment of contigs from SPS to a homologous sequence gives the primary sequence of a monoclonal antibody. Guthals et al. developed Meta-SPS and extended the de novo contig sequences to 100 amino acids with 97% accuracy.19 This group also explored de novo analysis of MS/MS triplets (CID/HCD/ETD) from overlapping peptides.20 Liu et al. developed Complete HomologyAssisted MS/MS Protein Sequencing (CHAMPS) for de novo sequencing. In CHAMPS, bottomup MS/MS was first employed to give de novo peptide sequences, and then these sequences were aligned to a homology protein sequence to find overlapping sequences, followed by final assembly to get the protein sequence. Liu et al. further developed top-down and bottom-up MSbased protein de novo sequencing (TBNovo) method, and got high sequence coverage and high sequence accuracy.21 Recently, Adrian Guthals and coworkers reported using semiautomated customized tools to sequence polyclonal antibodies. 13 One feature of pepsin in-membrane digestion is that we can control the peptic peptide sizes. The length of proteolytic peptides varies with the residence time of the protein solution in the membrane, i.e. shorter residence times generate longer peptides. We obtained middle-down sized peptides (4-9 kDa) in membrane-based digestions of antibodies when using a residence time of 3 msec. We also produced bottom-up sized peptides (0.3-2 kDa) when doing 3-sec digestion. Larger peptides give the arrangement of the small peptides, and small peptides give better 217 fragmentation performance compared to larger peptides. Both of the membrane digestion methods (short residence time and long residence time) gave 100% peptide coverage of the antibody light chain we examined, which may makes de novo antibody sequencing possible. Below I illustrate the strategy and results of the combination middle-down/bottom up procedure. I deconvoluted the 3 msec digestion MS file using Xtract with m/z from 600 to 1400, and minimum S/N set for 3. After analyzing the deconvoluted spectra, I found that four masses add (accounting for water in hydrolysis and protons) to give the mass of the light chain (M+H=24183.7). The four peptides are A 4204.1447, B 5234.5729, C 5649.8075, and D 9152.2347 (Numbers are monoisotopic M+H values). Peptides E 8177.0456, F 6892.4733, and D also add up to match the mass of the light chain. See Figure 5.3 below. Figure 5.3. Example of arranging peptides using relationships between their masses. The sum of two or more masses can give the mass of another peptide. Below I show relations among masses of peptides. Letters represent peptides from the 3-msec digestion, starred numbers or letters represent peptides from 30-msec digestion, and numbers represent peptides from the 3sec digestion. Regardless of a star, peptides with the same number or letter have the same sequence, e.g. 3=3*. 218 A+B+C+D=Light Chain (5-1) E+F+D=Light Chain (5-2) 1+G=E (5-3) 1+2+3+4=A (5-4) 1+2+3+5+6=A (5-5) 5+6=4 (5-6) 9+10=B (5-7) 11+12=B (5-8) 13+14+15=C (5-9) 16+17=D (5-10) 18+19=17 (5-11) 10+13+14+15=F (5-12) A+9=E (5-13) 2+3+4+9=G (5-14) 3*+4*=20* (5-15) 6*+9*=21* (5-16) 13*+14*=22* (5-17) 219 Note that the numbers of the peptides do not necessarily represent their order in the sequence. Next, we used these relations to try to order the peptides. Starting from (5-13) and (5-4), we have the order 1, 2, 3, 4, 9 or 9, 1, 2, 3, 4. The bold indicates this is a fixed position. We do not know the order of 1,2,3,4. From (5-14), we can deduce that 9 is connected to 2, 3, 4 and not 1. Thus, we have 1, 2, 3, 4, 9 or 9, 2, 3, 4, 1. (These peptides are not necessarily at the beginning of the sequence.) From (5-7), we deduce the sequence 1, 2, 3, 4, 9, 10 or 10, 9, 2, 3, 4, 1. Next, we look at (5-12), which shows we have 1, 2, 3, 4, 9, 10, 13, 14, 15 or 13, 14, 15, 10, 9, 2, 3, 4, 1. Also, looking at these sequences we either have ABC or CBA. Also, D could be at the beginning or the end. Continuing on, from (5-15), we learn that 3 and 4 are connected. From (5-16), 6 and 9 are connected. From (5-6), 6 is part of 4, so 9 is now connected to 4. Thus, we now have 1, 2, 3, 4, 9, 10, 13, 14, 15 or 13, 14, 15, 10, 9, 4, 3, 2, 1. We could easily fragment 1 to distinguish these possibilities. From (5-17), we see that 13 and 14 are connected. Assuming 1 is at the beginning (determined from fragmentation), we now have 1, 2, 3, 4, 9, 10, 13, 14, 15 or 1, 2, 3, 4, 9, 10, 14, 13, 15 or 1, 2, 3, 4, 9, 10, 15, 14, 13 or 1, 2, 3, 4, 9, 10, 15, 14, 13. If we compare fragmentation of 13 and 14 with fragmentation of C, we can figure out where 15 is. This will give us the alignment of the entire sequence of ABC as well as the smaller peptides. . We also have peptide D. Using the above equations, we can determine an order of 16, 17 or 16, 18, 19. We could also use what we know about the antibody. This is a mouse antibody. D is entirely in the constant region, so we already know its sequence and that it is on the C-terminus. Peptide 15 is also in the constant region, and we know it is connected to D. This finally leads us to 1, 2, 3, 4, 9, 10, 13, 14, 15, 16, 17. 220 We can align the peptic peptides of the antibody light chain just using the mass relations among the peptides. Then, future work should focus on whether we could perform de novo sequencing of the small peptides using CID, HCD and ETD MS/MS data. 5.2.4 Glycosidase-containing membrane for haptoglobin deglycosylation Glycosylation is an important protein PTM that enhances protein functional diversity and influences biological activities.22 Glycomics studies are challenging compared with proteomics and genomics due to the high diversity of sugars and their presence as an appendage on proteins. Current strategies for glycomics include analysis of glycopeptides generated from proteolysis of a glycoprotein, or analysis of glycans released from the glycoprotein using a glycosidase. Our research group developed several membrane reactors for protein digestion, protein purification and phosphorylated peptide enrichment.23-28 Fabrication of a glycosidase-containing membrane will further extend our work. Professor Radoslav Goldman from Georgetown University generously provided me an alkylated tryptic digest of haptoglobin as well as histidine-tagged N-Glycosidase F (PNGaseF). Our group previously developed functionalized membranes containing Ni2+ complexes for his-tagged protein capture.23 In this particular application, I followed the workflow of sequential deposition of polyelectrolyte (PAA/ polyethyleneimine /PAA) in a porous nylon membrane, followed by derivatization of PAA with nitrilotriacetate-Ni2+.23 I then immobilized His-tagged PNGaseF in these membranes through formation of the Ni2+-His tag complex and passed an alkylated tryptic digest of haptoglobin through this membrane reactor (3-sec residence time). Figure 5.4 shows the mass spectra of the haptoglobin digest before and after passing through the membrane. I 221 identified the glycopeptides by comparing the direct infusion MS data with the data in a recent paper.29 Figure 5.4. Comparison of mass spectra of haptoglobin tryptic peptides before (top) and after (bottom) passing the tryptic digest through a glycosidase-containing membrane (3-sec residence time). Not all of the identified peptides are labeled. AxGxSx is the name of a particular glycan structure where Ax is Man3GlcNAc2+ xGlcNAc, G is galactose and S is sialic acid. Theoretically, two tryptic glycopeptides, labeled T1 and T3, contain the glycosylation site. Figure 5.4 suggests that the membrane reactor removed the glycans on T1 because all the glycopeptide peaks disappear in the bottom mass spectrum. Another evidence is the appearance of a signal m/z of 894.1287 (3+) in the bottom mass spectrum, which corresponds to the peptide after glycan removal. However, glycopeptide T3 remained intact after passing through the membrane reactor, which indicates that the kinetics for removing glycans from different glycopeptides vary widely. Development of effective glycosidase-containing membranes will 222 require further work. Nevertheless, membrane-base glycan removal takes only 10 mins, compared with overnight for in-solution based protocols. 5.3 Summary of future work The proposed future work has four directions. The first is using protease-containing membranes to conduct limited digestion for analysis of protein higher order structures. Data interpretation is simple, and the differences in the signals in the mass spectra of two samples reveal the conformational change. The second direction is digestion of polyclonal antibodies. Preliminary results show successfully digestion of antibody mixtures. A LC-MS/MS workflow might be developed for optimization of the current method. The third direction is de novo sequencing of monoclonal antibodies. We proved that digestion of an antibody light chain at three different flow rates generates peptic peptides with overlapping sequences, and we could easily arrange the peptides using their mass relations. Future work should focus on choosing desired software for de novo sequencing of small peptic peptides. The last direction is the development of a glycosidase-containing membrane for glycan removal. This is an attractive application for membrane-based technology, and I am hoping to see more progresses in the near future. All the aforementioned applications can be performed by spin digestion. Compared with the syringepump digestion setup, the spin membrane has minimal dead volume, so possible non-specific adsorption of peptides on the digestion setup is no longer a problem. Also, multilayer membranes can be inserted into the spin column to gives spin-based membrane technology a myriad of possibilities for combination of processing and purification steps. 223 REFERENCES 224 REFERENCES (1) Vandermarliere, E.; Stes, E.; Gevaert, K.; Martens, L. Resolution of protein structure by mass spectrometry. Mass Spectrom. Rev. 2016, 35 (6), 653. (2) Tan, Y. J.; Wang, W. H.; Zheng, Y.; Dong, J.; Stefano, G.; Brandizzi, F.; Garavito, R. M.; Reid, G. E.; Bruening, M. L. Limited proteolysis via millisecond digestions in proteasemodified membranes. Anal. Chem. 2012, 84 (19), 8357. (3) Chait, B. T.; Cadene, M.; Olinares, P. D.; Rout, M. P.; Shi, Y. Revealing Higher Order Protein Structure Using Mass Spectrometry. J. Am. Soc. Mass. Spectrom. 2016, 27 (6), 952. (4) Wei, H.; Mo, J.; Tao, L.; Russell, R. J.; Tymiak, A. A.; Chen, G.; Iacob, R. E.; Engen, J. R. Hydrogen/deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications. Drug Discov. Today 2014, 19 (1), 95. (5) Huang, R. Y.; Chen, G. Higher order structure characterization of protein therapeutics by hydrogen/deuterium exchange mass spectrometry. Anal. Bioanal. Chem. 2014, 406 (26), 6541. (6) Morgan, C. R.; Engen, J. R. Investigating solution-phase protein structure and dynamics by hydrogen exchange mass spectrometry. Curr. Protoc. Protein Sci. 2009, Chapter 17, Unit 17 6 1. (7) Vahidi, S.; Stocks, B. B.; Konermann, L. Partially disordered proteins studied by ion mobility-mass spectrometry: implications for the preservation of solution phase structure in the gas phase. Anal. Chem. 2013, 85 (21), 10471. (8) Ecker, D. M.; Jones, S. D.; Levine, H. L. The therapeutic monoclonal antibody market. MAbs 2015, 7 (1), 9. (9) Maynard, J.; Georgiou, G. Antibody engineering. Annu. Rev. Biomed. Eng. 2000, 2, 339. (10) Fernandez, P.; Trenholme, A.; Abarca, K.; Griffin, M. P.; Hultquist, M.; Harris, B.; Losonsky, G. A.; Motavizumab Study, G. A phase 2, randomized, double-blind safety and pharmacokinetic assessment of respiratory syncytial virus (RSV) prophylaxis with motavizumab and palivizumab administered in the same season. BMC Pediatr. 2010, 10, 38. (11) Wang, X. Z.; Coljee, V. W.; Maynard, J. A. Back to the future: recombinant polyclonal antibody therapeutics. Curr. Opin. Chem. Eng. 2013, 2 (4), 405. 225 (12) Nahta, R.; Hung, M. C.; Esteva, F. J. The HER-2-targeting antibodies trastuzumab and pertuzumab synergistically inhibit the survival of breast cancer cells. Cancer Res. 2004, 64 (7), 2343. (13) Guthals, A.; Gan, Y.; Murray, L.; Chen, Y.; Stinson, J.; Nakamura, G. R.; Lill, J. R.; Sandoval, W.; Bandeira, N. De Novo MS/MS Sequencing of Native Human Antibodies. J. Proteome. Res. 2016, DOI:10.1021/acs.jproteome.6b00608. (14) Boutz, D. R.; Horton, A. P.; Wine, Y.; Lavinder, J. J.; Georgiou, G.; Marcotte, E. M. Proteomic identification of monoclonal antibodies from serum. Anal. Chem. 2014, 86 (10), 4758. (15) Johnson, R. S.; Biemann, K. The primary structure of thioredoxin from Chromatium vinosum determined by high-performance tandem mass spectrometry. Biochemistry 1987, 26 (5), 1209. (16) Hopper, S.; Johnson, R. S.; Vath, J. E.; Biemann, K. Glutaredoxin from rabbit bone marrow. Purification, characterization, and amino acid sequence determined by tandem mass spectrometry. J. Biol. Chem. 1989, 264 (34), 20438. (17) Bandeira, N.; Clauser, K. R.; Pevzner, P. A. Shotgun protein sequencing: assembly of peptide tandem mass spectra from mixtures of modified proteins. Mol. Cell. Proteomics 2007, 6 (7), 1123. (18) Bandeira, N.; Pham, V.; Pevzner, P.; Arnott, D.; Lill, J. R. Automated de novo protein sequencing of monoclonal antibodies. Nat. Biotechnol. 2008, 26 (12), 1336. (19) Guthals, A.; Clauser, K. R.; Bandeira, N. Shotgun protein sequencing with meta-contig assembly. Mol. Cell. Proteomics 2012, 11 (10), 1084. (20) Guthals, A.; Clauser, K. R.; Frank, A. M.; Bandeira, N. Sequencing-grade de novo analysis of MS/MS triplets (CID/HCD/ETD) from overlapping peptides. J. Proteome. Res. 2013, 12 (6), 2846. (21) Liu, X.; Dekker, L. J.; Wu, S.; Vanduijn, M. M.; Luider, T. M.; Tolic, N.; Kou, Q.; Dvorkin, M.; Alexandrova, S.; Vyatkina, K.et al. De novo protein sequencing by combining top-down and bottom-up tandem mass spectra. J. Proteome. Res. 2014, 13 (7), 3241. (22) Marino, K.; Bones, J.; Kattla, J. J.; Rudd, P. M. A systematic approach to protein glycosylation analysis: a path through the maze. Nat. Chem. Biol. 2010, 6 (10), 713. (23) Bhattacharjee, S.; Dong, J. L.; Ma, Y. D.; Hovde, S.; Geiger, J. H.; Baker, G. L.; Bruening, M. L. Formation of High-Capacity Protein-Adsorbing Membranes through Simple Adsorption of Poly(acrylic acid)-Containing Films at Low pH. Langmuir 2012, 28 (17), 6885. 226 (24) Tan, Y. J.; Wang, W. H.; Zheng, Y.; Dong, J. L.; Stefano, G.; Brandizzi, F.; Garavito, R. M.; Reid, G. E.; Bruening, M. L. Limited Proteolysis via Millisecond Digestions in Protease-Modified Membranes. Anal. Chem. 2012, 84 (19), 8357. (25) Tan, Y. J.; Sui, D. X.; Wang, W. H.; Kuo, M. H.; Reid, G. E.; Bruening, M. L. Phosphopeptide Enrichment with TiO2-Modified Membranes and Investigation of Tau Protein Phosphorylation. Anal. Chem. 2013, 85 (12), 5699. (26) Dong, J. L.; Bruening, M. L. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion. Annu. Rev. Anal. Chem. 2015, 8, 81. (27) Pang, Y. L.; Wang, W. H.; Reid, G. E.; Hunt, D. F.; Bruening, M. L. Pepsin-Containing Membranes for Controlled Monoclonal Antibody Digestion Prior to Mass Spectrometry Analysis. Anal. Chem. 2015, 87 (21), 10942. (28) Xu, F.; Wang, W. H.; Tan, Y. J.; Bruening, M. L. Facile Trypsin Immobilization in Polymeric Membranes for Rapid, Efficient Protein Digestion. Anal. Chem. 2010, 82 (24), 10045. (29) Pompach, P.; Brnakova, Z.; Sanda, M.; Wu, J.; Edwards, N.; Goldman, R. Site-specific Glycoforms of Haptoglobin in Liver Cirrhosis and Hepatocellular Carcinoma. Molecular & Cellular Proteomics 2013, 12 (5), 1281. 227