Protein surface accessible residues play an important role in protein folding, protein-protein interactions and protein-ligand binding. With the advantages in sensitivity, speed, and the capability of analyzing large/complex protein systems, mass spectrometry combined with protein labeling has found increasing utility for the characterization of protein surface. However, a common problem associated with the use of chemical labeling methods for mapping protein solvent accessible residues is that when a complicated peptide mixture resulting from a large protein or protein complex is analyzed, the modified peptides may be difficult to identify and characterize amongst the largely unmodified peptide population (i.e., the `needle in a haystack' problem). To address this challenge, an experimental strategy was developed involving the synthesis and application of a novel `fixed charge' sulfonium ion containing amine-specific protein modification reagent, S,S'-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS), coupled with capillary HPLC-electrospray (ESI)-MS, automated collision induced dissociation (CID)-MS/MS, and data dependent neutral loss mode MS3 in an ion trap mass spectrometer, to map the surface accessible lysine residues in a small model protein, Cellular Retinoic Acid Binding Protein II (CRABP II). After reaction with different reagent : protein ratios and digestion with Glu-C, modified peptides were selectively identified and the number of modifications within each peptide were determined by CID-MS/MS, via the exclusive neutral loss(es) of dimethylsulfide, independently of the amino acid composition and precursor ion charge state (i.e. proton mobility) of the peptide. The observation of these characteristic neutral losses were then used to automatically `trigger' the acquisition of an MS3 spectrum to allow the peptide sequence and the site(s) of modification to be characterized. Using this approach, the experimentally determined relative solvent accessibilities of the lysine residues were found to show good agreement with the known solution structure of CRABP II. With the initial success demonstrated on a model protein, the experimental strategy was extended to reveal the mechanisms corresponding to oxidation induced inactivation of calcineurin (CN), from a structural perspective. CN is a Ca2+/calmodulin (CaM) activated phosphatase that participates in a wide variety of physiological processes. CaN is also reported to be inactivated by H2O2- or superoxide-induced oxidation both in vivo and in vitro. However, the mechanism is still under debate. Here, the relative rates of H2O2 induced oxidation of methionine residues within CN were first determined using a multi enzyme digestion strategy coupled with analysis using capillary HPLC-ESI-MS and CID/ETD-MS/MS. Then the developed experimental strategy, i.e., combining protein modification by DMBNHS with data dependent multistage tandem mass spectrometry, was applied to characterize changes in CN conformation before and after oxidation. In addition, targeted ETD-MS/MS was used to characterize and quantify individual unmodified lysine residues from isomers of DMBNHS modified peptides containing multiple modifiable sites. The extent of DMBNHS modification of observed CN lysine residues was found to increase upon oxidation. More importantly, the methionine residues that were highly susceptible towards oxidation and lysine residues exhibited large increase in solvent accessibility upon oxidation all locate in CN functional domains that are involved in Ca2+/CaM binding regulated activation, thus indicating of a role for oxidation induced conformation change in CN as a possible cause of CN inactivation by inhibiting Ca2+/CaM regulated CN activations.
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