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This is to certify that the
thesis entitled

STUDIES ON THE FRAGMENTATION REACTIONS OF FIXED
CHARGE SULFONIUM ION CONTAINING PEPTIDES BY TANDEM
MASS SPECTROMETRY

presented by

MAHASILU AMUNUGAMA

has been accepted towards fulfillment
of the requirements for the

MS. degree in CHEMISTRY

 

 

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2/05 p:lClRC/DaleDue.indd-p.1

STUDIES ON THE FRAGMENTATION REACTIONS OF FD(ED CHARGE
SULFONIUM ION CONTAINING PEPTIDES BY TANDEM MASS
SPECTROMETRY

BY

MAHASILU AMUNUGAMA

A THESIS

Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of

MASTER OF SCIENCE

Department of Chemistry

2006

ABSTRACT
STUDIES ON THE FRAGMENTATION REACTIONS OF FIXED CHARGE
SULFONIUM ION CONTAINING PEPT IDES BY TANDEM MASS
SPECTROMETRY
BY
MAHASILU AMUNUGAMA
In order to enable the development of improved tandem mass spectrometry based
methods for selective proteome analysis, the mechanisms, product ion structures and
other factors influencing the gas-phase fragmentation reactions of methionine side chain
derivatized 'fixed-charge' phenacyl sulfonium ion containing peptide ions have been
examined. Dissociation of these peptide ions results in exclusive loss of the derivatized
side chain, thereby enabling their selective identification. The resultant product ion(s) are
then subjected to further dissociation to obtain sequence information for subsequent
protein identification. Molecular orbital calculations (at the BBLYP/6-31+G** level of
theory) performed on a simple peptide model, together with experimental evidence
obtained by multistage dissociation of a regioselectively deuterated methionine
derivatized sulfonium ion containing tryptic peptide, indicate that the initial
fragmentation occurs via 8N2 reactions resulting in the formation of cyclic five- and six-
membered hydrofuran and oxazine product ions. Further, molecular orbital calculations
and experimental evidence obtained from various para-substituted phenacyl sulfonium
ion peptide ions have determined that the ratio of neutral versus charged losses of the
derivatized side chain exhibits a linear dependence on the proton affinity of the side chain

fragmentation product, as well as the proton mobility of the peptide product ions.

ACKNOWLEDGMENTS

No dissertation is ever successful due to one persons efforts, and certainly this one
was no different. It would never have become produced without the help and suggestions
of many people. Dr. Gavin E. Reid has been a great source of guidance and motivation.
He is more than an advisor. His leadership in both personal and scientific endeavors has
been an inspiration to me throughout my graduate career, and I am grateful for having
had the opportunity to pursue my master’s degree under his supervision. Special thanks
are also extended to Dr. Merlin Bruening and Dr. Gary J. Blanchard who are on my thesis
committee. I would also like to thank Dr. Kade D. Roberts for all the support, advice and
encouragement he provided me throughout my graduate studies. I am grateful to James
Sierakowski, who is a wonderful friend, for his help in many ways to make my research
successful. I am also deeply indebted to my dear friends Jennifer Marie Froelich and
Gwynyth Scherperel for their continuous support and encouragement. They were always
with me during the good, difficult and distressing times in my graduate career. Special
thank also goes to all the group members of the Reid research group for their support. I
especially thank the free education system in Sri Lanka without which I would have
never been able to come to graduate school. I am eternally grateful to my parents who,
though living oceans apart, have shown me how to live life with grace and compassion.

Finally, I am thankful to all who have made this journey both possible and pleasurable.

TABLE OF CONTENTS

LIST OF FIGURES ................................................................. viii
LIST OF TABLES ................................................................. xi
LISTOFSCHEMES............................ ................................... xii
1. CHAPTER ONE: Introduction .................................................. 1
1.1 Proteomics .................................................................. 1
1.2 Mass Spectrometry ......................................................... 1
1.2.1 Electrospray Ionization (ESI) ........................................ 2
1.2.2 Matrix-assisted laser desorption ionization (MALDI) ........... 4
1.2.3 Mass Analyzers ........................................................ 5
1.2.3.1 Quadrupole Mass Analyzers .................................... 5
1.2.3.2 The Quadrupole Ion Trap Mass Analyzer .................... 10

1.3 Current Approaches Employed for Proton Identification and

Characterization by Mass spectrometry ................................. 13
1.3.1 Database Search Algorithms for Protein Identification ......... 15
1.4 Understanding and Controlling Peptide Fragmentation Reactions... 17

1.4.1 Mechanisms for the Fragmentation of Protonated Peptide Ions... 18
1.4.2 Controlling Peptide Fragmentations ................................... 22
1.4.2.1 N-terminal Fixed Charge Derivatization and Fragmentation 22

1.5. Side Chain Fixed Charge Derivatization and Fragmentation ............... 23

1.6 Aims of this Thesis ......................................................... 27

iv

2. CHAPTER TWO: Experimental .................................................
2.1 Materials .....................................................................
2.2 Synthesis of (D, L)—, (D)- and (d4-D, L)-Fmoc-Methionine ..........
2.3 Synthesis of 4-Bromoacetylbenzoic- acid .................................
2.4 Synthesis of Methyl-4—bromoacetylbenzoate ............................
2.5 Synthesis of 4-Methylphenacy1bromide ..................................
2.6 Synthesis of 4-Ethylphenacylbromide......................................
2.7 Side chain Fixed-Charge Derivatization of Methionine-Containing

Peptides ......................................................................
2.8 Mass Spectrometry .........................................................
2.9 Computational Methods ...................................................
2.9.1 Computational Methods Employed for Mechanistic Studies.
2.9.2 Computational Methods Employed for Proton Affinity Calculations

2.9.3 Proton Affinity Calculations .........................................

3. CHAPTER THREE: Mechanisms for the Selective Gas-Phase
Fragmentation Reactions of Methionine Side Chain Fixed Charge
Sulfonium Ion containing Peptides ................................................

3.1 Introduction ..................................................................
3.2 Molecular Orbital Calculations to Examine Mechanisms for the Loss
of CH3SR from Methionine Fixed Charge Sulfonium Ion Containing

Peptides ..........................................................................

28

28

29

29

3O

30

3O

31

31

33

33

33

34

37

37

38

3.3 Potential Ring Opening of the Five and Six Membered Cyclic
Product Ions via Intramolecular Proton Transfer Reactions ..............
3.4 Obtaining Experimental Evidence for the Mechanisms Responsible
for the Loss of CH3SR from Methionine Fixed Charge Sulfonium
Ion Containing Peptides. . . . . . . . . . . . .. .....................................
3.4.1 Regioselective Deuterium Labeling Experiments to Examine
Mechanisms for the Side Chain Fragmentation Reactions of
Methionine Fixed Charge Containing Peptide Ions .................
3.4.2 Multistage Tandem Mass Spectrometry Experiments to
Examine the Structure(s) of the Product Ions Formed by Side
Chain Fragmentation of Methionine Fixed Charge Containing
Peptides ............................................................................... . ......

3.5 Conclusion ...................................................................

4. CHAPTER FOUR: Substituent Effects on the Fragmentation Reactions of
Methionine Side Chain Fixed Charge Phenacylsulfonium Ion Containing
Peptides .......................................................................................................

4.1 Introduction ..................................................................

4.2 Proton Affinity Calculations ...............................................

4.3 Determining the Influence of Side Chain Fragment Proton Affinities
on the Relative Abundance of Neutral versus Charged Side Chain
Losses from Methionine Fixed Charge Containing Peptide

Ions .....................................................................................................

vi

47

53

53

57

62

63

63

65

4.4 Conclusions and Future Directions ................................................. . 78

REFERENCES ........................................................................ 8O

vii

LIST OF FIGURES

FIGURE PAGE

1.1 Components of a mass spectrometer .................................................................. 3

1.2 Stability areas as a function of U and V for positive ions with different masses
where ml <m2 < m3< m. We can scan all ions by changing U linearly as a
function of V along a straight operating line ............................................................. 9

1.3 Typical Mathieu stability diagram for quadrupole ion trap. The larger balls
represent high mass ions whereas smaller balls represent low mass ions ................. 12

1.4 Dehmelt pseudo well potential D (eV) as a function qz. Higher mass ions for a
given V value have lower Dehmelt trapping energies .............................................. 14

3.1 Optimized precursor, transition state and product ion structures (at the
B3LYP/ 6-31+G** + ZPVE level of theory) for the loss of CH3SCH3 from the
simple model peptide N-acetyl methionine-N-methyl amide, due to a neighboring
group participation reaction involving (A) the C-terminal carbonyl group to form
a 5 membered hydrofuran product ion (Pathway l), or (B) the N-terminal
carbonyl group to form a 6 membered oxazine product ion (Pathway 2). The
transition state and product ion structures for ring opening of the product ions
formed from Pathways 1 and 2, involving intramolecular proton transfer (Bl-H) to
either the N-terminal carbonyl group (Pathway 1’ in A) or the C-terminal
carbonyl group (Pathway 2’ in B) to yield protonated vinyl glycine product ions,
are also shown. The energies of all molecules are given with respect to structure
A ................................................................................................................................ 44

3.2 Optimized precursor, transition state and product ion structures (at the
B3LYP/ 6-31+G** + ZPVE level of theory) for the loss of CH3SCH3 from the
simple model peptide N-acetyl methionine-N-methyl amide due to intramolecular
proton (BI-H) transfer to (A) the C-terrninal carbonyl group (Pathway 3) or (B)
the N-terminal carbonyl group (Pathway 4) to yield protonated vinyl glycine. The
energies of all molecules are given with respect to structure A in Figure 3.1 .......... 46

3.3 Optimized precursor, transition state and product ion structures (at the
B3LYP/ 6-31+G** + ZPVE level of theory) for the loss of CH3SCH3 from the
simple model peptide N-acetyl methionine-N-methyl amide involving the
alternate intramolecular proton (Bg-H) transfer to (A) the C-terminal carbonyl

viii

group (Pathway 3-2) or (B) the N -terminal carbonyl group (Pathway 4-2) to yield
protonated vinyl glycine. The energies of all molecules are given with respect to
structure A in Figure 3.1 ............................................................................................

3.4 Optimized precursor, transition state and product ion structures (at the
B3LYP/ 6-31+G** + ZPVE level of theory) for ring opening of the product ions
formed from Pathways 1 and 2, involving the alternate intramolecular proton ([32-
H) transfer to either the N-terminal carbonyl group (Pathway 1’ in A) or the C-
terrninal carbonyl group (Pathway 2’ in B) to yield protonated vinyl glycine
product ions. The energies of all molecules are given with respect to structure A...

3.5. Summary of the predicted low energy transition state barriers (at the
B3LYP/6-31+G** +ZPVE level of theory) for the dissociation of methionine side
chain fixed charge containing sulfonium ion peptides .............................................

3.6 Linear ion trap CID MS" analysis of a methionine side chain phenacyl
sulfonium ion fixed charge derivative of GAIL(d4.-M)GAILK. (A) CID MS/MS
product ion spectrum of the [Mi’+H]2+ ion. (B) CID MS/MS product ion spectrum
of the [M++2H]”’ ion. (C) CID M33 product ion spectrum of [M++H-CH3SR]2+
ion from Figure 3.6A ................................................................................................

3.7 Multistage (MS/MS, MS3 and MS”) tandem mass spectrometry analysis of
GAILM(R)GAILK (where R=CH2COC6H5) and GAILAGAILK. (A) CID M33
product ion spectrum of the [M++H-CH3SR]2+ ion from GAILM(R)GAILK. (B)
CID MS4 product ion spectrum of the ya product ion from Figure 3.7A. (C) CID
MS4 product ion spectrum of the y7 product ion from Figure 3.7A. (D) CID
MS/MS product ion spectrum of the [M-I-2H]2+ ion of GAILAGAILK (E) CID
MS3 product ion spectrum of the y, product ion from Figure 3.7D. (F) CID MS3
product ion spectrum of the y7 product ion from Figure 3.7D .................................

4.1 Optimized low energy structures at the B3LYP/ 6-31+G** + ZPVE level of
theory for the neutral and protonated products of the para-substituted
methylphenacylsulfide derivatives employed for proton affinity calculations .........

4.2 Linear ion trap CID MS/MS analysis of methionine side chain fixed charge
phenacylsulfonium ions of GAILMGAILK. (A) CID MS/MS product ion
spectrum of the [M+] ion. (B) CID MS/MS product ion spectrum of the [M"+H]+2
ion. (C) CID MS/MS product ion spectrum of the [MJ’+2H]+3 ion ...........................

4.3 Linear ion trap CID MS/MS analysis of methionine side chain fixed charge
phenacylsulfonium ions of VTMGHFDNFGR. (A) CID MS/MS product ion

ix

48

50

52

55

66

68

spectrum of the [M++H]2+ ion. (B) CID MS/MS product ion spectrum of the
[M++2H]3+ ion. (C) CID MS/MS product ion spectrum of the [M++3H]4+ ion .........

4.4 Linear ion trap CID MS/MS analysis of methionine side chain fixed charge
phenacylsulfonium ions of VTMAHFWNFGK. (A) CID MS/MS product ion
spectrum of the [M"+H]2+ ion. (B) CID MS/MS product ion spectrum of the
[M++2H]3+ ion. (C) CID MS/MS product ion spectrum of the [M++3H]4+ ion ........

4.5 Linear ion trap CID MS/MS analysis of methionine side chain fixed charge
para-substituted phenacylsulfonium ions of GAILMGAILK. (A) CID MS/MS
product ion spectrum of the para-benzoic acid phenacylsulfonium [M"+2H]3+ ion.
(B) CID MS/MS product ion spectrum of the para-methylbenzoate
phenacylsulfonium [M++2H]3+ ion. (C) CID MS/MS product ion spectrum of the
para-methyl phenacylsulfonium [M++2H]3+ ion, and (D) CID MS/MS product ion
spectrum of the para-ethyl phenacylsulfonium [M"+2H]3+ ion .................................

4.6 Linear ion trap CID MS/MS analysis of methionine side chain fixed charge
para-substituted phenacylsulfonium ions of VTMGHFDNFGR. (A) CID MS/MS
product ion spectrum of the para-benzoic acid phenacylsulfonium [Mir-3H]4+ ion.
(B) CID MS/MS product ion spectrum of the para-methylbenzoate
phenacylsulfonium [M++3H]4+ ion. (C) CID MS/MS product ion spectrum of the
para-methyl phenacylsulfonium [M++3H]4+ ion, and (D) CID MS/MS product ion
spectrum of the para-ethyl phenacylsulfonium [MW-3H]4+ ion .................................

4.7 Linear ion trap CID MS/MS analysis of methionine side chain fixed charge
para-substituted phenacylsulfonium ions of VTMAHFWNFGK. (A) CID MS/MS
product ion spectrum of the para-benzoic acid phenacylsulfonium [MW-3H]4+ ion.
(B) CID MS/MS product ion spectrum of the para-methylbenzoate
phenacylsulfonium [M’”+3H]4+ ion. (C) CID MS/MS product ion spectrum of the
para—methyl phenacylsulfonium [MW-3H]4+ ion, and (D) CID MS/MS product ion
spectrum of the para-ethyl phenacylsulfonium [M"+3H]4+ ion ................................

4.8. Plot of ln[-CH3SR / (-(CH3SR+H+) + CH3SR+H+)] versus calculated proton
affinity for the phenacylsulfide derivatives of (A) the [M+2H]2+ side chain
cleavage product ion of GAILMGAILK (B) the [M-i-3H]3+ side chain cleavage
product ion of VTMGHFDNFGR and (C) the [M+3H]3+ side chain cleavage
product ion of VTMAHFW N FGK ...........................................................................

7O

71

73

74

75

77

LIST OF TABLES

TABLE PAGE

3.1 Total Energies (Emmi), Zero point Vibrational Energies (ZPVE) and relative
energies (Eml) computed for the precursor ions, transition state and product ion
structures associated with each reaction pathway at the B3LYP/6-3 I+G** level of
theory ......................................................................................................................... 41

4.1 Total energies (Emmi), zero point vibrational energies (ZPVE), vibrational
energies (EVibmfional) and proton affinities computed at the B3LYP/6-31+G** level
of theory for the para-substituted methylphenacylsulfide derivatives ...................... 67

xi

LIST OF SCHEMES

SCHEME PAGE
1.1 Nomenclature peptide fragment ions .................................................................... 19
1.2 Mechanism for formation of b-type, y-type, a-type and immonium ions ............ 21

1.3 Fixed charge derivatization and selective fragmentation of methionine
sulfonium ion derivatized peptide ions ...................................................................... 25

1.4 Potential mechanisms for the selective fragmentation of methionine sulfonium
ion derivatized peptide ions .......................................................................................

26
2.1 The structures of sulfonium ion derivatization reagents ..................................... 32
2.2 The structures of neutral and protonated para-substituted S-methyl phenacyl
derivatives .................................................................................................................. 36
3.1 Possible reaction mechanisms to account for the neutral loss of CH3SR from
methionine side chain fixed charge containing peptide ions ..................................... 39
3.2 Potential intermolecular deuterium transfer reactions for the charged loss of
CH3SR+D+ from methionine side chain fixed charge containing peptide ions ........ 56
3.3 Structures of (A) the five membered hydrofuran product ion formed via the
loss of CH3SR from GAILM(R)GAILK, (B) the six membered oxazine product
ion formed via the loss of CH3SR from GAILM(R)GAILK, and (C)
GAILAGAILK. The amide bond cleavage sites for formation of the y5, y6 and y7
product ions in each case are indicated ...................................................................... 59

xii

CHAPTER ONE

INTRODUCTION

1.1 Proteomics

The rapid completion of the human genome sequence [1] has enabled research
efforts aimed at determining the unknown functional roles of the proteins encoded by
these genes. However, in contrast to the static genome, the proteome is dynamic. Proteins
are continuously synthesized and degraded in order to carry out the functions of the cell.
Proteins can often be post-translationally modified, and interact with other proteins in
such a way that they can function as a part of a large protein complex. The studies of
proteins are important because it is believed that a disease can arise due to altered protein
expression. Thus, changes in protein expression or modification state may be used in
medicine as specific biomarkers for the early diagnosis of disease. Therefore, the
systematic large scale analysis of protein expression under normal and perturbed cellular
conditions, such as stressed, diseased, or drugged states may lead to the development of
novel therapeutic drugs [2]. The major goal of proteomic studies is the comprehensive
separation, identification, characterization and quantitative analysis of all the gene
products, i.e., proteins, expressed by a particular cell or tissue type at a given time,
together with the systematic characterization of their specific localization, modification

state and functional interactions.

1.2 Mass Spectrometry

Mass spectrometry, which measures an intrinsic property of a molecule (i.e., mass
to charge) is one of the most important analytical techniques whose beginning dates back
to the start of the last century. Figure 1.1 shows the main components of a mass
spectrometer. The mass spectrometer consists of an ion source from which the charged
ions are generated, a mass analyzer that measures the mass to charge ratio (m/z) of the
ionized analytes and a detector that convert the ion flux into a proportional electric
current. A data system then records the magnitude of the electrical signal as a function of
m/z which is then shown as a mass spectrum. In the early 19805, fast atom bombardment
(FAB) mass spectrometry was employed to identify and characterize biological
molecules [3, 4, 5]. Since the late 1980’s, the development of the electrospray ionization
(E81) [6] and matrix-assisted laser desorption ionization (MALDI) [7] soft ionization
techniques, coupled with the development of improved mass analyzers yielding better
sensitivity and higher resolution, has led to mass spectrometry becoming an essential tool

for proteomics research.

1.2.1 Electrospray Ionization (ESI)

Fenn, er. al. first introduced the electrospray ionization (ESI) technique in the late
1980’s [6]. A liquid containing the analyte is pumped through a small diameter needle to
which a high voltage is applied to produce an electrospray containing small micrometer
sized droplets. These charged droplets rapidly evaporate in the presence of high
temperature or a sheath gas, and undergo a series of coulombic fission events to yield
gas-phase ions according to the charge residue [8] or the ion evaporation models [9, 10].

The most significant advantage of electrospray ionization technique is that it is operated

..........................................................................................

longFormation Ion Separation Ion Detection

Sample Ionization Mass
[ Introduction ]C>[ Source ]C>[ Analyzer jd LDeteotor jé

Vacuum

Pump
Data
Handling [ Data System J

11

Data | i 1
Output l 7 g l

Mass
spectrum

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1.1 Components of a mass spectrometer (adapted from “What is Mass

Spectrometry”. www.asms.org).

under atmospheric pressure, enabling a wide range of compounds including proteins and
peptides that are sufficiently polar to allow attachment of a charge to be analyzed. ESI
can be operated in either an infusion mode or in combination with high performance
liquid chromatography (HPLC). In the infusion mode, the analyte is dissolved in a
suitable solvent (typically 1:1 methanolzwater) and introduced to the ESI capillary via an
injection pump at flow rates as low as few hundred nanoliters per minute. When on-line
HPLC is employed, the analytes are introduced to the ESI source as they elute from the
chromatographic column. Hence, sample clean up, concentration and separation of the
analytes can be achieved in a single step. A striking feature of ESI is the ability to form a
series of pseudomolecular ion charge states, including both singly and multiply charged
ions. The extent of multiple charging is influenced by factors such as the composition and
pH of the electrospray solvent, as well as the chemical nature of the analyte. These
multiple charge states can be used to provide independent verification of the mass, and
allow large molecules such as proteins and peptides to be observed using mass analysers

with limited m/z charge ranges.

1.2.2 Matrix-assisted laser desorption ionization (MALDI)

Karas and Hillenkarnp developed matrix-assisted laser desorption ionization
(MALDI) technique in the late 19805 [7]. MALDI is achieved in two steps. First the
analyte is mixed with a solvent containing small organic molecules (matrix) that has a
strong absorption at the wavelength of the laser employed for ionization, then allowed to
dry on a metal substrate. Second, the resulting solid is placed under vacuum and

irradiated by nanosecond laser pulses for a short duration. This generates a rapid heating

of the matrix crystals and sublimation of matrix and analyte molecules into the gas phase.
Ionization can occur during any time of this process, however, the exact mechanism by
which ionization takes place is not yet fully understood [11]. Typically, singly charged

pseudomolecular ions are observed by MALDI.

1.2.3 Mass Analyzers

The mass analyzer is one of the most important components of the mass
spectrometer. Once the ions have been produced, the mass analyzer separates ions
according to their mass to charge (m/z) ratio. Key analytical figures of merit in
proteomics include resolution, sensitivity and mass accuracy, which are all dependant on
the performance of the mass analyzer. There are four basic types of mass analyzers
currently used in proteomics. They are (i) quadrupole (ii) quadrupole ion trap, (iii) time
of flight (TOF) and (iv) Fourier transform ion cyclotron resonance (FT- ICR) analyzers.
The quadrupole and quadrupole ion trap mass analyzers are described in more detail

below.

1.2.3.1 Quadrupole Mass Analyzers

Quadrupole mass analyzers are made using four parallel hyperbolic or cylindrical
rods, upon which is imposed a variable amplitude oscillating AC (RF) voltage
superimposed with a variable DC voltage to separate ions according to their m/z ratios.

The potentials applied to opposite sets of rods ((D0) are given by the following equations.

(D0 = + (U-Vcos wt) and - (D0 = - (U-Vcos wt)

where U is the amplitude of the DC potential, V is the amplitude of the AC potential, (1) =

27w is the angular frequency (v is the frequency of the radio frequency) and t is time.

The force on the ions in the x and y directions of the quadrupole rods are given by

 

dzx 3(1)
Fx—m?——ze$ .................... (I)
2
3(1)
Fy=m dtzy =-ze3y- ..................... (2)

Where (I) is a function of (D0

2 2
_ 2 2 2_(x -y )*(U-Vcosax)
(DU—(Dow —y )/r0 — 2 ........... (3)

’0

 

After differentiating and rearranging this leads to the following equations

 

 

2
$.55]. 2282 (U ~Vcosax)x = O ................... (4)
dt rm
0
d2 y 2ze
2 — 2(U-Vcosa)t)y=0 ..................... (5)
dt er

In this equation, x and y are the distances of the ion from the center of the quadrupoles in
the x and y directions, respectively. U and V are variables. As long as the values of x and
y do not reach re, the distance between the center and the quadrupole, the trajectories of
the ions are stable. These two differential equations are similar to the Mathieu equation

written below

dzu
(1:2

+ (au — 2qu cos 2§)u = 0 ..................... (6)
In equation (6), ‘u’ is similar to either x or y in equation (4) and (5), where f = %

Therefore, the previously determined solutions for au and qu from the Mathieu equation

can be used to obtain solutions for U and V.

(I -a — a - 82€U
u" x"- y‘—
mwzroz
q “q _ q _ 4zeV
u— x“ y‘—
mwzroz

After rearranging these equations, we obtain the following equations used to derive the

Operating parameters for the quadrupole mass analyzer.

22

 

 

ma) r0
= a ..................... (7)
28e
ma)2 r02
= q“ ... ................... (8)
z4e

In these solutions, r0 and a) are constants for a given quadrupole. Therefore, for a given
m/z, r0, e and (D are constant. A plot of U and V gives the stability areas for different
masses where ions have a stable trajectory through the quadrupole. Figure 1.2 shows the
stability areas for positive ions with increasing m/z values from m; to m. To obtain a
mass spectrum, we can scan the different ions by changing U linearly as a function of V
at a constant UN ratio along a straight line near the apex of the stability diagram. When a
given UN ratio is selected near the apex, only one m/z ion corresponding to that UN
ratio is stable, whereas other ions are unstable and are lost. Thus, the higher the slope of
scanning line, the higher the resolution. If the slope of the scanning line is low, a range of
m/z ions will be stable at a given UN value, resulting in lower resolution since the
stability arrears overlap each other [12].

Quadrupole mass spectrometers have been constructed to perform tandem mass
spectrometry (MS/MS) by coupling three quadrupole mass analyzers together in series. In
a triple quadrupole mass spectrometer, the first and the third quadrupoles are used to scan
ions while the second is used as a collision cell. A product ion scan mode tandem mass
spectrum can be obtained by selecting a precursor ion of interest using the first

quadrupole and by fragmenting it in the second quadrupole while scanning the third

Scanning at constant UN

<tr

 

Figure 1.2 Stability areas as a function of U and V for positive ions with different masses
where ml <m2 < m3< m. We can scan all ions by changing U linearly as a function of V

along a straight operating line. (Reproduced and modified from the reference [12]).

quadrupole for product ions. Similarly, precursor ion scan, neutral loss scan and selected

reaction monitoring experiments can be performed [12].

1.2.3.2 The Quadrupole Ion Trap Mass Analyzer

In the three dimensional ion trap, a variable amplitude radio frequency is applied
to the ring electrode to create a three dimensional quadrupole electric field to trap ions at
the center of the trap. Typically, the ion trap is filled by an inert gas (such as helium) to a
pressure of about 1 mTorr. Ion collisions with the inert gas decreases the kinetic energy
of the ions and help to contract their trajectories quickly toward the center of the trap
under the influence of the oscillating radio frequency field. The solution of the Mathieu
equation can also be used to describe the theory of ion motion in three dimensional ion
traps. Since a DC potential is not required for ion storage, the solution for all of Mathieu

equation is equal to zero whereas qz is given by following equations,

 

 

4zeV -
u— 2 2 ....................... (9)
mroa)
8zeV
qu—-2q — 2 2 2 ................... (10)

where r0 and 20 which are constant for a given ion trap, are the distances from the centre

of the trap to the ring electrode and end cap electrodes, respectively, V is the amplitude of
the RF potential, (1) is the angular velocity of the RF potential which is a constant at a

fixed frequency. Equation (9) represents the q value for an ‘ideal’ quadrupole ion trap,

10

whereas equation (10) represents the stretched quadrupole ion trap geometry from which

the well characterized problem of mass shift can be overcome [l3]. Ions are stable in the

ion trap as long as their trajectories do not reach the distances r0 and zo. Figure 1.3 shows

the stability diagram for the 3D quadrupole ion trap. According to the equation (9) and
(10), q is inversely proportional to the m/z value and directly proportional to V. In this
figure, larger balls represent high m/z ions at low q values and small balls represent low
m/z ions at high q values. Since there is no DC voltage applied, the ion trap is operated
along the V (q) axis. When V is increased, ions move towards higher q values. Once ions
exceed a q value of 0.908 at the boundary of the Mathieu stability diagram, the ions
trajectory becomes unstable and is ejected. Some percentage of these ions reaches the
detector. However, in order to obtain improved mass analysis performance, a
supplementary (auxiliary) radio frequency is typically applied to the end cap electrodes to
enable resonance ejection. In this mode, when V is increased, ions again move towards
higher q values. However, when the ions reach a q value corresponding to the applied
resonance ejection RF value below the normal instability q value as shown in Figure 1.3,
ions are rapidly destabilized and ejected from the ion trap. The supplementary RF may
also be used for ion excitation during MS/MS.

The main advantage of the ion trap is the ability to perform multistage tandem
mass spectrometry (MSn) experiments. This can be achieved by isolation of an ion of
interest by applying a high amplitude broadband resonance ejection supplementary RF
signal to the end caps to eject all ions except the ion of interest. Then the isolated ion is
subjected to a low amplitude single frequency supplementary RF signal. The ion

undergoes energetic collisions with the inert gas present in the trap, and subsequently

ll

RF Ejection

Stable Ions

 

 

 

 

V

v or m.)

Figure 1.3 Typical stability diagram for quadrupole ion trap. The larger balls represent

high mass ions whereas smaller balls represent the low mass ions. (Reproduced and

modified from reference [12])

12

undergoes dissociation. This fragmentation is performed at a relatively low V (q) value to
reduce the low mass cut off. However, if the V value is too low during fragmentation, it
may be difficult to apply enough energy for ion fragmentation to occur prior to ion
ejection, due to the lower Dehmelt potentials at low V. The Dehmelt potential curve is
shown in Figure 1.4. The energy ERE in the Figure 1.4 represents the energy that should
be provided for that ion by resonant excitation to eject the ion from the trap. Other
advantages of ion traps are sensitivity, robustness and their relative inexpense. The main
disadvantage of the ion trap is the low mass accuracy due to the limited number of ions
that can be accumulated before space charge effects distort the ion motions. The recently
developed linear two-dimensional (2D) quadrupole ion trap can be used to overcome
most of these disadvantages. The linear ion trap generates a two dimensional electric field
and thus ions can enter the trap more easily. Ions are trapped by a combination of RF
applied to the quadrupole rods, and a static DC potential applied to the electrodes situated
at both end of quadrupoles. The linear ion trap also has higher ion storage capacity and
exhibits less space charge effects [14,15]. Alexander Makarov et a! very recently
developed a new orbitrap mass analyser from which high mass accuracy up to parts per

million and high mass resolution can be obtained [16,17,18,19].

1.3 Current Approaches Employed for Protein Identification and Characterization
by Mass Spectrometry
Prior to mass spectrometry, protein mixtures are typically partially resolved by column

chromatography, affinity chromatography or one- or two-dimensional gel electrophoresis.

13

 

 

 

 

—l
V

Figure 1.4 Dehmelt pseudo well potential D (eV) as a function qz. Higher mass ions for a
given V value have lower Dehmelt trapping energies. (Reproduced and modified from

reference [12]).

14

Once a protein of interest is adequately purified, it is then enzymatically digested
(usually with trypsin) to form a series of peptides. These peptides are then introduced into
the mass spectrometer to obtain mass spectra. Although these masses may provide a
‘peptide mass fingerprint’ that can be compared to the theoretically expected peptides in
a database to identify the protein of interest, the intact masses alone may not be sufficient
for unambiguous protein identification or characterization. This is a particular issue for
the identification and characterization of post translationally modified proteins, or for the
identification of proteins present in more complex unresolved mixtures. Therefore,
tandem mass spectrometry (MS/MS) experiments are commonly performed to obtain
information regarding the amino acid. Protein identification is then achieved by database

analysis as described below.

1.3.1 Database Search Algorithms for Protein Identification

Several database search algorithms, such as Sequest [20] and Mascot [21], have
been developed for protein identification using mass spectrometry derived data. These
algorithms calculate a score for how well an experimental product ion tandem mass
spectrum agrees with a theoretical product ion spectrum generated by the search engine.
The algorithms first theoretically cleave all the proteins in the database into peptides
according to the experimental enzyme cleavage specificity, then extract all the peptides
from the database whose masses correspond to the mass of the experimentally observed
peptide, within a given mass tolerance. The presence of post translational or chemical
modification to certain amino acids may be accounted for in this step. Then, the matching

peptides are theoretically fragmented to give the masses of the b- and y- type ions, and

15

compared to the experimentally observed product ion masses. Peptides are accepted as
genuine hits if they fulfill criteria such correct enzymatic cleavage specificity, a score
above a pre-defined cut off value, and the absence of other peptides that match with
similar scores [22, 23].

The success of the database search approaches outlined above requires that the
experimental peptide sequence must be contained within the sequence database. Thus,
when the protein of interest is not in the database, it is necessary to determine its identity
by de novo peptide sequence analysis [24]. Furthermore, the success of the algorithms
also depends on their ability to accurately predict the types and abundances of the product
ions that will be generated by MS/MS. The database search algorithms typically only
consider product ions formed by amide bond cleavage. However, most spectra also
contain product ion formed via the loss of water, ammonia, carbon monoxide, cleavage of
side chains, or internal fragmentations. Furthermore, some amino acid residues within a
peptide sequence lead to unusual cleavage pathways which are not adequately accounted
for by current algorithms. Phosphorylation, oxidation, acetylation, methylation,
glycosylation, ubiquitination and lipid modifications are some of the most common post
translational modifications (PTMs) [25]. Post-translational modifications change the
mass of the attached amino acid within the peptide. Although the Sequest and Mascot
algorithms have been extended to account for the changes in peptide mass occurring as a
result of these post translational modifications [23, 26], they typically do not account for
changes in the product ion spectra induced by these modifications. Oxidation of
methionine containing proteins, as a result of post translational modification or sample

handling, may result in the characteristic ‘non-sequence’ neutral loss of methane sulfenic

l6

acid (CH3SOH, 64 Da) [27]. Reid et. al. have recently studied the fragmentation reactions
of a series of methionine sulfoxide containing peptides. Under mobile-proton conditions
(the mobile proton model is described in section 1.4.1), fragmentation of the protonated
precursor ions resulted in non-specific fragmentations along the peptide backbone,
whereas dissociation of the same peptides under non-mobile proton conditions resulted in
exclusive loss of CH3SOH at the methionine sulfoxide side chain via a cis 1,2 elimination
mechanism (a “charge remote” mechanism). Phosphorylation of serine and threonine also
often leads to ‘non-sequence’ fragmentation reactions involving the neutral loss of
phosphoric acid (H3P04, 98 Ba) [28] under ‘non-mobile proton’ conditions. In these
cases, the non-sequence derived product ions may dominate the spectrum, suppressing
the formation of sequence ions [28, 29]. Therefore, as the dominant ‘non-sequence’
product ion fragmentation pathways are not taken into consideration by the search
algorithms, when the required sequence ions are in low abundance or absent, low

database search scores may result.

1.4. Understanding and Controlling Peptide Fragmentation Reactions

The limitations of the search algorithms described above are mainly due to the
lack of understanding of the factors that effect the gas phase fragmentation reactions of
peptide ions upon MS/MS. Therefore, determining the mechanisms and other factors
influencing the dissociation behavior of peptide ions is a key to increasing the accuracy
of search algorithms employed for protein identification, or for the development of
methods for controlling and directing the fragmentation of peptide ions toward the

formation of analytically useful product ions.

17

1.4.1 Mechanisms for the Fragmentation of Protonated Peptide Ions

Scheme 1.1 shows the generally accepted peptide fragment ion nomenclature. The
ions a, b, and c represent products retaining the charge on the N-terminal, whereas ions x,
y and 2 denote products retaining the charge on the C-terminal. b- and y-type ions
originating via the cleavage of C-N amide bonds along the peptide backbone are typically
the dominant product ions produced under the low energy collision induced dissociation
conditions employed in triple quadrupole and quadrupole ion trap mass spectrometers.

The fragmentation of amide bonds under low energy activation conditions are
influenced by the ability of an ionizing proton to migrate from the initial site of
protonation (the side chains of basic amino acids, or the N-terminal amino group) along
the peptide backbone to an amide bond, where localization of the proton promotes bond
cleavage. This has been called the ‘mobile proton model’ by Dongre at. e1. [30]. The
initial site of protonation is determined by the relative proton affinities of the various
functional groups contained within the peptide. Experimental mass spectrometry
approaches to determining proton affinities have been described [31,32]. Alternatively,
proton affinities may be determined by theoretical molecular orbital modeling
calculations [33, 34]. Proton mobility can be divided into 3 categories; “non mobile”
when the total number of protons S the number of Arg residues, “mobile” when the total
number of protons > than the total number of basic residues (i.e. combined number of
Arg, Lys and His), or “partially mobile” when the total number of basic residues 2 the
total number of protons 2 number of Arg residues. [35, 27]. Under ‘mobile’ or partially-
mobile’ proton conditions, migration of an ionizing proton may result in localization at an

amide nitrogen along the peptide backbone.

18

a, b1 c1 az b2 c2 a3 b3 c3
—l m ‘-

R (i R"

=O/
\
\
z:
/
\
n—
/
/
\
20/
\
Z:
/
0:0

/ \

HZN OH

o
:q
:1:
o
73

 

 

 

 

 

 

 

 

 

X3 3’3 23 x2 Y2 22 xi 3’1 21

Scheme 1.1 Nomenclature for peptide fragment ions.

19

It is generally considered that fragmentation of the amide bond then occurs by
nucleophilic attack from an adjacent amide carbonyl to yield complementary b- and/or y-
type product ions, as shown in Scheme 1.2. Oxazolone b-type product ions may then lose
CO to form a—type ions [36]. The losses of small neutral molecule such as water or
ammonia, via similar mechanisms can also be observed [37]. Note that although Scheme
1.2 shows the amide bond cleavage reaction proceeding via an amide nitrogen protonated
precursor, it has been shown using theoretical ab initio and semi-empirical molecular
orbital calculations that protonation of the carbonyl oxygen of the amide bond is
thermodynamically preferred over protonation of the amide nitrogen in peptide ions
[38,39,40,41,42]. Thus, fragmentation via an amide carbonyl oxygen protonation
precursor should also be considered as a viable dissociation pathway.

The presence of additional basic residues in peptide ions can lead to localization
of the ionizing protons at their side chains (i.e., resulting in ‘non-mobile’ proton
conditions). Under these conditions, higher energies are required for proton mobilization,
and as a result, alternative dissociation reactions driven by “charged remote”
fragmentation mechanisms may predominate, such as those involving enhanced
‘sequence’ cleavages at the C-terrninal of aspartic acid residues [35,43] or enhanced
‘non-sequence’ cleavages at the side chains of methionine sulfoxide residues [27],

suppressing other ‘sequence’ ion formation.

20

I

OH

\
l o
- o
E
2>P_
o=xi
\
z
"3
n
/

 

 

path b
H+ transfer

 

y2 ion
-HNCO
‘CO -mcnco
R
A i .
+
HzN (If/ +\/ HZNVR
O immonium ion
an ion

Scheme 1.2 Mechanisms for the formation of b-type, y-type, a-type and immonium ions.

21

1.4.2 Controlling Peptide Fragmentation Reactions

Understanding the mechanisms for peptide fragmentation has been a key step
toward developing methods for controlling or directing peptide fragmentation towards the
formation of analytically useful product ions from which peptides or proteins may be
more readily identified and/or characterized. A number of different methods for the
control of peptide fragmentation, involving the introduction of a fixed charge to the

peptide, have been developed.

1.4.2.1 N-terminal Fixed Charge Derivatization and Fragmentation

Fixed charge derivatization strategies for peptides have been used previously in
conjunction with MS/MS for sequencing applications. However, most prior work has
been limited to derivatization of the N- and C-termini, and has largely been focused on
directing fragmentation toward formation of a series of amide backbone cleavage
‘sequence’ ions [44,45]. Watson and co-workers have demonstrated that N-terminal
peptide derivatization with tris(2,4,6-trymethoxyphenyl) phosphonium (TMPP)+-
Ac_SC6F5 bromide results in the formation of a well controlled series of N-terminal a-
type product ion series under high energy fragmentation conditions [46]. Adamczyk et.
al. have used a similar reagent [tris(2,4,6-trymethoxyphenyl) phosphonium] acetic acid
N-hydroxysuccinimide ester (TMPP+-Ac_OSU) to generate a series of N-terminal b-type
ions accompanied by a less intense a-type product ion series under low energy
fragmentation conditions in an ion trap mass spectrometer [47]. It is believed that the
formation of N-terminal b- and a—type ions are due to a ‘charge remote’ mechanism

involving a shift of the amide hydrogen to the C-terminal carbonyl carbon of the amide

22

bond [48]. Wysocki and co-workers have demonstrated that the selective gas phase
cleavage of the amide bond C-terminal to Aspartic acid residues maybe controlled using
a ¢3P+CH2C(=O)- [<D=2,4,6—trimethoxyphenyl) reagent introduced to the N—terminal of

the peptide [49].

1.5 Side Chain Fixed Charge Derivatization and Fragmentation

Reid et. al. have recently described a novel method for the selective identification
of peptide ions, via the derivatization and selective fragmentation of peptide ions
containing certain side-chain functional groups [50,51]. In contrast to the fixed charge
derivatization methods described above, the MS/MS dissociation of peptide ions
containing these fixed charge derivatives are directed toward exclusive formation of a
single characteristic side chain cleavage product ion at the site of the fixed charge. Thus,
the fixed charge containing peptide ions may be readily identified from complex mixtures
for example, by selective neutral loss scan mode MS/MS methods. Incorporation of 'light'
and ‘heavy’ isotopically encoded labels into the fixed-charge derivatives also facilitates
the application of this MS/MS method to the quantitative analysis of differential protein
expression. This approach thereby offers the potential for significantly improved
sensitivity and selectivity for the identification and quantitative analysis of peptides or
proteins containing certain structural features, without the requirement for extensive
fractionation or otherwise enrichment from a complex mixture prior to analysis.

The fixed charge derivatization and selective fragmentation reactions of peptides
containing methionine were described in the above mentioned work, via the initial

formation of a side chain phenacylsulfonium ion (Scheme 1.3). Mechanisms to account

23

for the loss of CH3SCH2COC6H5 from the methionine containing peptide ions following
low energy CID MS/MS were proposed (Scheme 1.4), involving either of the amide
carbonyl groups adjacent to the side chain in intramolecular E2 proton transfer
elimination (pathway 1) or 8N2 elimination (pathway 2) reactions. These mechanisms
would result in elimination of the side chain to yield acyclic 3-amino-1-butenoic acid
(vinyl glycine) or five or six membered cyclic containing peptide product ions,
respectively. It was further proposed that ring opening of the cyclic product ions via an
intramolecular proton transfer reaction (pathway 2a), could occur to yield the acyclic
vinyl glycine containing product. However, extensive studies to determine the operating
mechanisms, and the influence of the resultant product ion structures on subsequent
fragmentation reactions, were not performed. Thus, given that further characterization of
the sequence of the identified methionine containing peptides may be achieved by
subjecting the initial MS/MS product ions to multistage MS/MS (M83) in a quadrupole
ion trap mass spectrometer, or by energy resolved 'pseudo‘ M83 in a triple quadrupole
mass spectrometer, it is important to determine the mechanisms by which the neutral loss
occurs and to determine the structures of the resultant product ions.

It was also noted in the above mentioned work that while the fragmentation of side chain
fixed charge methionine containing peptides were observed to undergo exclusive
dissociation at the amino acid side chain, independently of the amino acid composition
and precursor ion charge state (i.e., proton mobility), the loss of the side chain fragment
as a neutral versus protonated species was observed to vary depending on the proton

mobility of the precursor ion.

24

H3C\ H3c\§t/\

S C
l W. (I;
('31: (i) BrCH2C0C6H5 CH2 (iii) CID
—> —> [M +nn]"‘+”+
0 CH2 (in Ms 0 CH2 (:iijscnzcocfiii5
ll 1.. s t! in i
,J-r \N/ ‘C/ my JJ-f \N/ ‘C/ ‘11”
H II H II
0 0
[M] [M+nH+CH2COC6H5]("+”+

Scheme 1.3 Fixed charge derivatization and selective fragmentation of methionine

sulfonium ion derivatized peptide ions.

25

O CH
II | +
Pathway 1 H I
HN
H C / “LL.
3 \+/\C
1I I
H, C\ q 0 Pathway 2a
H‘H Pathway l
Pathway 2 '0' .3 Pathway 2
C\ n CH /O°
’\/\I\/ \§/ \C/ CH n H
/ 2. I
CH \Pathway 2a
H ILHZ ? f‘ I o.
H |
”NH—L,

Scheme 1.4 Potential mechanisms for the selective fragmentation of methionine

sulfonium ion derivatized peptide ions.

26

Thus, if neutral loss or precursor ion scan mode MS/MS and MS3 methods are to be
employed for the comprehensive identification of side chain fixed charge peptide ions,

the factors influencing this loss must be more fully understood.

1.6 Aims of this Thesis

The aims of this thesis are:

l. to identify the mechanisms and product ion structures associated with the neutral
loss of CH3SR from the side chains of methionine fixed charge sulfonium ion
containing peptides, and;

2. to determine the influence of para substituents on the ratio of charged versus
neutral loss product ions formed by dissociation of phenacylbromide alkylated

fixed charge sulfonium ion containing methionine peptides.

27

CHAPTER TWO

Experimental

2.1 Materials

Unless stated otherwise all reagents were analytical reagent (AR) grade and used
as supplied without further purification. The synthetic tryptic peptides GAILMGAEK
and GAILAGAILK were obtained from Auspep (Melbourne, Vic.). Poly(4-
vinylpyridiniam tribromide) and Phenacylbromide (Compound 1 in scheme 2.1) were
purchased from Fluka (Switzerland). Fmoc-chloride, methanol (HPLC grade), 4-
Acetylbenzoic acid, Ethylacetate, Acetyl Chloride, 4-Acety1benzoic acid, D,L-
Methionine and D-Methionine were purchased from Sigma-Aldrich (St. Louis, MO,
USA). d4-D,L-Methionine was purchased from CDN Isotopes (Canada). Glacial acetic
acid (ACS grade), P-Dioxane, Magnesium Sulfate, Sodium Carbonate and Sodium
bicarbanate were purchased from Spectrum Chemicals (Gardena, CA, USA). Acetonitrile
(HPLC grade) was purchased from OmniSolv (Gibbstown, NJ, USA). Hydrochloric acid
and hexane (ACS grade) were purchased from Columbus Chemical Industries
(Columbus, WI, USA). Diethyl Ether and Bromine were purchased from Jade Scientific
(Canton, Michigan, USA). Ethyl acetate was purchased from Mallinckrodt Chemicals
(Phillipsburg, NJ, USA). 4-Ethy1acetophenone and 4-Methylacetophenone were
purchased from Acros Organics (New Jersey, USA). All solutions were prepared using
deionized water purified by a Barnstead nanopure diamond purification system

(Dubuque, Iowa, USA).

28

2.2 Synthesis of (D, L)-, (D) - and (d4-D, L)-Fmoc-Methionine

D-Methionine (100 mg), D,L-methionine (100 mg), or d4-D,L-Methionine (100
mg), were dissolved in 5 ml of 10% N32CO3. Then 2.5 mL of P-Dioxane was added and
finally, 170 mg of Fmoc-chloride dissolved in 5 mL of P-Dioxane was added dropwise
while stirring at 0°C. The reaction was allowed to proceed for one hour at 0°C and then
continued for 24 hours at room temperature. The resulting mixture was poured onto 75
ml of ice-water and chloroformate was removed by extracting twice with diethyl-ether
(40 mL). The pH of the resulted aqueous layer was adjusted to 2 by adding conc. HCl at
0°C. Fmoc-Methionine was extracted twice into diethyl-ether (40mL) and dried with
MgSO4. The solvent was evaporated under reduced pressure and Fmoc-Methionine was
recrystallized with EtOAc-hexane. The GAIL-(D)-MGAILK, GAIL-(DL)-MGAILK and '
GAIL-(d4-DL)-MGAILK peptides were then synthesized by Syn Pep Company (Dublin,

CA, USA).

2.3 Synthesis of 4-Bromoacetylbenzoic acid (Compound 2 in Scheme 2.1)
4-Acetylbenzoic acid (503 111, 3.37 mmol) was dissolved in acetic acid (20 mL)
and the reaction mixture was heated at 70 °C until dissolution was achieved. The reaction
solution was then cooled to room temperature and Bromine (190 uL, 3.7 mmol) was
added in one portion with stirring which was continued over night at room temperature.
The reaction mixture was then filtered, the precipitate washed with water and dissolved in
Ethylacetate (20 mL). The insoluble material was filtered and the filtrate washed with
water (20 mL x 1). The Ethylacetate was then removed under a gentle stream of N2, then

completely dried under high vacuum to yield a white solid.

29

2.4 Synthesis of Methyl-4-bromoacetylbenzoate (Compound 3 in Scheme 2.1)

Acetyl chloride (1.6 mL, 2 mol) was added drop wise to Methanol (10 mL) with
cooling and stirring in an ice-bath. 4’-Acetylbenzoic acid (328 mg, 2 mmol) was then
added in one portion and the reaction mixture was left to stir at room temperature for 3
days. The solvent was allowed to evaporate off overnight at room temperature to yield a
white/yellow solid, which was then collected and dried under vacuum. The solid was then
dissolved in Acetic acid (10 mL) and Bromine (190 uL, 3.7 mmol) was added in one
portion with stirring at room temperature, which was continued over night. The reaction
solution was then diluted with H20 (50 mL) and extracted with Diethyl ether (3 x 20
mL). The combined organic extracts were washed with 0.1M NaHCO3 (2 x 20 mL) and
H20 (1 x 20 mL), then dried over MgSO4 and concentrated invacuo to give a white solid

which was dried under high vacuum.

2.5 Synthesis of 4-Methylphenacylbromide (Compound 4 in Scheme 2.1)
4'-Methylacetophenone (0.5g, 3.7 mmol) was added to poly(4-vinylpyridinium

tribromide) (1.86 g , ~3 meq Br3' lg resin) in 20 mL Methanol. The reaction mixture was

then stirred at room temperature for 4 hours. The reaction mixture was filtered, and the

methanol was evaporated off under a gentle stream of N; to yield a white solid.

2.6 Synthesis of 4-Ethylphenacylbromide (Compound 5 in Scheme 2.1)

4-Ethylacetophenone was dissolved in acetic acid and bromine was added in one

portion with stirring at room temperature which was continued over night. The reaction

30

solution went from a dark brown/orange color to a light yellow color over this time. The
reaction solution was diluted with H20 (50mL) and extracted with Diethyl ether (3 x
20mL) then H20 (1 x 20mL) then dried over MgSO4 and concentrated under reduced

pressure to give a dark yellow liquid.

2.7 Side Chain F ixed-Charge Derivatization of Methionine-Containing Peptides

Side chain fixed-charge sulfonium ion derivatives of synthetic methionine-
containing model “tryptic” peptides were produced by the addition of 10 [.tL of a 1M
solution of alkylating reagent (Compounds 1-5 in Scheme 2.1) to 100 11g of either
GAILMGAILK, GAIL-(D)—MGAILK, GAE-(D,L)-MGAILK, GAIL-(d4-D,L)-
MGAILK, VTMAHFWNFGK or VTMGHFDNFGR dissolved in 100 uL of aqueous
20% HOAc containing 30% CH3CN. The reactions were allowed to proceed for 24 h at
room temperature after which the samples were diluted and introduced to the mass

spectrometer without further purification.

2.8 Mass Spectrometry

Peptides were introduced to a linear quadrupole ion trap mass spectrometer
(Thermo model LTQ, San Jose, CA) by electrospray ionization (ESI). Samples, (0.02
mg/mL) dissolved in 50% MeOH, 1% AcOH were introduced into the mass spectrometer
at 0.5 [LL/min. The spray voltage was set at 1.8 kV. The heated capillary temperature was
200°C. CID MS/MS and MS3 experiments were performed on monoisotopically mass

selected ions using standard isolation and excitation procedures.

31

O
0
Br
OH
O
0
Br
0 3
\
O
O
O
Br\/n\©\/
Scheme 2.1 The structures of sulfonium ion derivatization reagents.

32

2.9 Computational Methods
2.9.1 Computational Methods Employed for Mechanistic Studies

The model peptide N-acetyl methionine (S,S—dimethy1 sulfonium)-N-methyl
amide (CH3CONHCH(CHzCHzS+(CH3)2)CONHCH3) was used for computational
calculations. Low energy transition state structures related to the possible mechanisms for
dissociation of the methionine sulfonium ion side chain, were initially found at the PM3
semi empirical level of theory, followed by further optimization at the B3LYP level of
theory using the 6-31+G(d,p) basis set using the GAUSSIAN 98 (version 5.2) molecular
modeling package. Intrinsic reaction coordinate (IRC) searches were then performed,
followed by geometry optimizations to locate the appropriate reactant and product ion
structures associated with each transition state. All optimized structures were subjected to
harmonic vibrational frequency analysis and visualized using the computer package
Gauss View 2.1 to determine the nature of the stationary points. Zero point energies were

obtained from harmonic frequency calculations without scaling.

2.9.2 Computational Methods Employed for Proton Affinity Calculations

Full conformational searches on neutral and protonated para—substituted S-methyl
phenacyl derivatives (Compounds 6-15 in Scheme 2.2) were initially performed at the
PM3 semi empirical level of theory, followed by further optimization of low energy
structures at the B3LYP level of theory using the 6-31 + G (d,p) basis set using the
GAUSSIAN 98 (version 5.2) molecular modeling package. All optimized structures were

subjected to harmonic vibrational frequency analysis and visualized using the computer

33

package Gauss View 2.1 to determine the nature of the stationary points. Zero-points

energies were obtained from harmonic frequency calculations without scaling.

2.9.3 Proton Affinity Calculations
Consider the following equation for the molecule M and its protonated structure,

MH+.

M + H+ —-) MH” (Eq.1)
The proton affinity (PA) of a molecule M can be calculated according to the negative of
the enthalpy (AH) of Eq. 1 via the equations below, where ARH is the enthalpy of the
reaction.

PA = -ARH°293 (Eq. 2)

The enthalpy of the reaction can be written in terms of the translational (AtE), rotational

(AB), and vibrational (AVE) energy of the species,

ARH°298 = -AxE + A(AEI.298) + A(AE,,398) + MAE/.298) + APV (Eq. 3)

AXE = Eclcc(MH+)- Eclcc(M) + ZPVE(MH+)— ZPVE(M) (Eq. 4)

, where APV = -RT, Eclcc is the electronic energy, and ZPVE is the zero point vibrational

energy correction (i.e., the difference in translational, rotational and vibrational energy

34

from OK to 298K). The value of the translational energy A(AE(,293) = 3/2RT and the

rotational energy A(AE,,293) = 0- Eclec(MH+)r Eelec(M)r AEv.298 (MW), Ev.298(M) and
ZPVE values may all be obtained from the output of the structural optimizations
described above. Thus, the proton affinity of a molecule can be calculated using the final

equation below.

ARH 298 = Eelcc(MH+)' Eclec(M) + ZPVE(MH+)‘ ZPVE(M) + AEv,298 (MH+)' Ev,298(M) '

5/2RT (Eq. 4)

35

o +0H
\S \s
fir 6 /il\©\'( 11
OH OH
0 o
o +OH
\S \s
7 12
O\ o\
0 o
o +0H
NCO ° EKG ‘3
0 ton
\SJKQ 9 \S)K©\ 14
O +on
\S/u\©\/ 1o \SJ\©\/ 15

Scheme 2.2 The structures of neutral and protonated para—substituted S-methyl phenacyl

derivatives.

36

CHAPTER THREE
Mechanisms for the Selective Gas-phase Fragmentation Reactions of Methionine

Side Chain Fixed Charge Sulfonium Ion Containing Peptides

3.1 Introduction

The gas-phase fragmentation reactions of peptide ions containing a
phenacylsulfonium fixed charge on the side chain of methionine residues have recently
been examined in order to develop a novel strategy to selectively identify methionine
containing proteins by tandem mass spectrometry [50]. It was shown that dissociation of
these fixed charge derivatives under low energy CID MS/MS conditions results in
exclusive loss of the derivatized side chain, with the formation of a single characteristic
side chain cleavage product ion (Scheme 1.3 in Chapter 1). It was further demonstrated
that the characteristic loss of the fixed charge sulfonium ion methionine side chain could
be used to selectively identify methionine containing peptides from within complex
mixtures by selective neutral loss scan mode MS/MS methods. Although several
mechanistic possibilities involving intramolecular E2 or 8N2 elimination reactions
(Scheme 1.4 in Chapter 1) were proposed for these selective fragmentation reactions in
the above mentioned work, extensive studies to determine the operating mechanisms, and
the influence of the resultant product ion structures on subsequent fragmentation
reactions, were not performed. Determining these mechanisms and product ion structures
is of particular importance, as characterization of the amino acid sequence of the
methionine containing peptides identified via the diagnostic loss of the side chain may

potentially be achieved by further subjecting the initial MS/MS product ions to multistage

37

MS/MS (MS3) in a quadrupole ion trap mass spectrometer, or by energy resolved 'pseudo'
MS3 in a triple quadrupole mass spectrometer. Here, a combination of transition state
calculations at the B3LYP/6-31+G** level of theory performed on a simple peptide
model, as well as experimental studies based on the multistage dissociation of a
regioselectively deuterated methionine derivatized sulfonium ion containing tryptic
peptide, have been carried out to obtain evidence to support the proposed SN2 reaction

mechanism.

3.2 Molecular Orbital Calculations to Examine Mechanisms for the Loss of CH3SR
from Methionine Fixed Charge Sulfonium Ion Containing Peptides.

As shown in Scheme 3.1, the neutral loss of CH3SR from the side chains of
methionine fixed charge containing peptide ions could occur via several different
mechanistic pathways. Pathways 1 and 2 of Scheme 3.1 shows neighboring group
participation reactions involving either nucleophilic attack from the carbonyl group of the
C-terminal amide bond, or the carbonyl group of the N-terminal amide bond,
respectively. These reactions pathways would result in the formation of protonated five
membered hydrofuran or six membered oxazine product ions, respectively. Alternatively,
pathways 3 and 4 of Scheme 3.1 show E2 elimination reactions involving intramolecular
proton transfer from the [3 methyl group of the methionine fixed charge containing side
chain to the N or C-terrninal amide carbonyl oxygens to yield protonated acyclic 3-
amino-l-butenoic acid (vinyl glycine) product ions. Furthermore, the five membered
hydrofuran and the six membered oxazine product ions formed from Pathways l and 2 in

Scheme 3.1 could potentially undergo intramolecular proton transfer ring opening

38

 

 

/

14.3 kcal mol ' 312:0 Pathway 1' cl)" 1 27.0 kcal mol'1
Pathway 1/\C .. C< 45.0 kcal mol" /C\§ C/O Pathway 3
\i‘l C\\ " l

HN;\ HN\
\t/R \g/
Pathway 2 S) Pathway l Pathway" 4 )
ii)? ICI,‘ 0)" HPathway 3
/c<‘. 2° / 0 5%
H H
.1; .13
Pathway 2 $6.31"? Pathway 2' [if I Pathway 4
c " o
16.2 kcal mol ‘ / \ c; 45. 3 kcal mol ' /C \N /<|3l/OH 28. 3 kcal mol '
.13 ..i

Scheme 3.1. Possible reaction mechanisms to account for the neutral loss of CH3SR from

methionine side chain fixed charge containing peptide ions.

39

reactions to yield the vinyl glycine product ions (Pathways 1' and 2' in Scheme 3.],
respectively).

To determine which of the mechanistic possibilities described in Scheme 3.1 are
energetically feasible, a series of molecular orbital calculations were performed to
determine the transition state barriers associated with each reaction pathway. Potential
transition state structures for each of the possible mechanisms shown in Scheme 3.1 were
initially examined at the PM3 semi empirical level of theory, using a simple peptide
model, N-acetyl methionine-N-methyl amide, containing a fixed charge on the thioether
side chain, CH3CONHCH(CH2CH2S+(CH3)2)CONHCH3. Low energy conformers were
then re-optimized at the B3LYP/6-31+G** density functional level of theory.
Vibrational frequency analysis was then performed to determine the nature of the
optimized stationary point structure. Then, intrinsic reaction coordinate searches were
performed, followed by geometry optimization at the same level of theory to locate the
appropriate precursor and product ion structures associated with each transition state
structure. The total energies, zero point vibrational energies (ZPVE) and relative energies
(total energies + ZPVE, relative to structure (A)) obtained for each of the optimized low
energy transition state structures, as well as those of the optimized precursor,
intermediate and product ion structures are given in Table 3.1.

Structure TSl in Figure 3.1(A) shows the predicted low energy transition state
structure for the loss of CH3SCH3 to form a protonated five membered hydrofuran
product ion via an SN2 neighboring group participation reaction involving the amide bond
C-terminal to the methionine residue. After optimizing this structure, IRC calculations

followed by geometry optimizations were performed to locate the precursor ion (A) and

40

Table 3.1 Total energies (Emmi), zero point vibrational energies (ZPVE) and relative
energies (E...) computed for the precursor ions, transition states, intermediate product

ions and product ion structures associated with each reaction pathway at the B3LYP/6-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

31+G** level of theory.
Structure Emmi (Hartree) ZPVE (Kcal mol'r) Em. (kcal mol'])“‘5

A -1012.103755 178.01581 0.0
TS] -1012.080944 176.84406 +14.3

B -1012.1 12208 178.01406 -5.3

C -534. 1521621 177.46909 +0.9
T52 -534.081918 124.64946 +450
D -534.1330484 128.06278 +12.9

E -1012. 100463 177.68801 +2.1
T33 -1012.07791 176.74105 +16.2

F - 1012.1 14764 178.32422 -6.9

G -534. 150836 130.07242 +1.7
T54 -534.0813041 124.88532 +453
H -534.l325096 127.84273 +13.2

I -1012.096112 [77.93925 +4.8
T55 -1012.0607l3 173.96721 +27.0

J -1012.092994 [76.09255 +6.7
K -534. 1324999 127.8488 +13.2

 

 

 

 

41

 

Table 3.1 (continued)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

L -1012.101371 177.91885 +1.5
T86 ~1012.058067 173.63621 +28.7
M -1012.0951 1 175.66895 +5.4

-534. 1330465 128.06403 +12.9
O -1012. 107568 178.4326 -2.3
T87 -1012.058023 173.90797 +28.7
P -1012.09213 175.65843 +7.3
Q -534. 1306281 128.0657 +14.4
-1012.09593 178.13605 +4.9
T88 4012058689 173.865 +28.3
S -1012.088737 175.95845 +9.4
T -534. 1281226 127.75565 +16.0
U -534. 1478423 129.97671 +3.6
T89 -534.0731 1 13 12495592 +505
V -534. 1404751 127. 10571 +8.2
T810 -534.0641064 124.99521 +56.l
W -534. 1249397 127.93632 +18.0
8(CH3)2 -477.9502026 47.51316

 

 

 

 

42

a13ml: total energy + (ZPVE). bEnergy relative to structure A.

 

the intermediate product ion (B) associated with this reaction coordinate. The relative
energies of the transition state and the product ion were then calculated with respect to
the energy of the precursor ion. The product ion (C) was then obtained by geometry
optimization following the removal of CH38CH3 from structure (B). The relative energy
of (C) was obtained by summing the energy of the product ion (C) and the energy of the
geometry optimized CH3SCH3. Structure (T83) in Figure 3.1(B) represents the low
energy transition state structure for the neutral loss of CH3SCH3 via a neighboring group
participation reaction involving the amide bond N-terminal to the methionine residue to
form a protonated six membered oxazine product ion. After performing IRC calculation
on transition state (T83), the optimized precursor ion, (E) the intermediate (F) and the
product ion (G) were obtained as mentioned above. The energies of all these structures
are given with respect to the energy of structure (A).

According to Figure 3.1(A), the predicted transition state barrier for pathway 1, to
yield the cyclic protonated five membered hydrofuran ring, is 14.3 kcal mol'1 with
respect to the energy of structure (A), whereas the transition state barrier to form the six
membered oxazine ring via pathway 2 is predicted to be 16.2 kcal mol'1 relative to the
energy of the structure (A). The predicted transition state barriers for pathways 1 and 2
(structures (T81) and (T83) in Figure 3.1) are therefore relatively comparable. The
slightly lower energy of transition state (T81) is likely due to better hydrogen bonding
between the carbonyl oxygen and the amide nitrogen on the N- and C-terminal amide
bonds (1.93 A in (T8 1) versus 2.08 A in (TS3)). The predicted energy difference between 1

these transition states is not considered significant enough to suggest the formation of one

43

1.85 A \ "’8‘
.4
Pathway 1 2 2 A 2. 49 A 85A Pathway 2 2. 05 A 2.48 A
2. 27 “A 2.08 A

   

2.02 A 1.93 A
(A) (T81) (T83)
0.0 kcal mol“1 14.3 kcal mol'l 7 2.1 kcaEl) mol ' 16. 2 kcal mol l
1.48 A 3 02 A\- 1 47A
1. 75 A 1.99 A
1 74 A
(G)
0.9 kcaCl )'mol ’ -.5 3 kcal mol ' 1.7 kcal mol‘l 6. 9 kcal mol '
lPathway l' l Pathway 2'
1.27 A 1.24 A 1-34 A
2.19 A
139 A—. 1.4.49 A
1.87 A 1.70 A
(T82) (D) (T84) (H)
45.0 kcal mol" 12.9 kcal mol'l 45.3 kcal mol’I 13. 2 kcal mol’l

Figure 3.1 Optimized precursor, transition state and product ion structures (at the B3 LYP/
6-31+G** + ZPVE level of theory) for the loss of CH3SCH3 from the simple model
peptide N-acetyl methionine-N-methyl amide, due to a neighboring group participation
reaction involving (A) the C-terminal carbonyl group to form a 5 membered hydrofuran
product ion (Pathway 1), or (B) the N-terminal carbonyl group to form a 6 membered
oxazine product ion (Pathway 2). The transition state and product ion structures for ring
opening of the product ions formed from Pathways 1 and 2, involving intramolecular
proton transfer (Br-H) to either the N-terminal carbonyl group (Pathway 1’ in A) or the C-
terminal carbonyl group (Pathway 2’ in B) to yield protonated vinyl glycine product ions,
are also shown. The energies of all molecules are given with respect to structure A.

Images in this thesis/dissertation are presented in color

44

particular product over another for the loss of CH3SCH3 via either reaction pathways 1 or
2. Thus, both the five and six membered rings could potentially be formed.

Figure 3.2(A) shows the predicted low energy transition state structure (T85)
found for the E2 elimination reaction involving intramolecular proton transfer of one of
the two hydrogens from the B methyl grOup of the methionine side chain to the C-
terrninal carbonyl group to subsequently yield a vinyl glycine product ion. IRC
calculations were again performed to obtain the appropriate precursor and intermediate
product ions (D and (J), respectively. The structure of product ion (K) was obtained by
optimization of intermediate structure (J) after removal of CH38CH3, as mentioned
above. The energies of these structures are given with respect to the energy of structure
(A) in Figure 3.1. Figure 3.2(B) shows the predicted low energy transition state structure
(T86) associated with the E2 elimination reaction involving intramolecular proton
transfer of one of the two hydrogens from the [3 methyl group of the methionine side
chain to the N-terminal carbonyl group. The energies of this transition state relative to
structure (A) in Figure 3.1 was predicted to be 28.7 kcal mol'l. Furthermore, as there are
two hydrogens on the [3 methyl group in the methionine side chain, transition-state
calculations predicting the barriers to the E2 elimination reactions involving the alternate
hydrogens to those shown in Figure 3.2 were also determined. The structures associated
with these transition states (structure (T87) in pathway 3-2 and structure (T88) in
pathway 4-2) are shown in Figures 3.3. The relative energies of these transition states
were predicted to be 28.7 and 28.3 kcal mol", respectively. Thus, the four E2 elimination

reaction pathways all have comparable activation barriers.

45

      

2.30 A ‘ LSSQ
Pathwa 3 1-4' A Pathwa 4 1.22
2.23 A —yo r 1.24 A 3.30 A 2.28 A __Z
2'07 1.93 A

11) (T55) (L) (T56)

4.8 kcal mol'l 27.0 kcal mol " 1.5 kcal mol'| 28.7 kcal mol "
.49 A
+ 5((‘H312 o——— L73 A +S(CH3)2 *— (é
1.7011 “87A LssA

(K) U) (N) (M)

13.2 kcal mol " 6.7 kcal mol'l 12.9 kcal mol " 5.4 kcal rnol‘l

Figure 3.2 Optimized precursor, transition state and product ion structures (at the B3 LYP/
6-31+G** + ZPVE level of theory) for the loss of CH3SCH3 from the simple model
peptide N-acetyl methionine-N-methyl amide due to intramolecular proton (Br-H) transfer
to (A) the C-terminal carbonyl group (Pathway 3) or (B) the N-terminal carbonyl group
(Pathway 4) to yield protonated vinyl glycine. The energies of all molecules are given

with respect to structure A in Figure 3.1.

46

Pathways 1 and 2 in Figure 3.1 can be compared with pathways 3, 3-2, 4 and 4-2
in Figures 3.2 and 3.3 to predict the favorable mechanistic pathway for the neutral loss of
CH38CH3. The transition states associated with the neighboring group participation
processes (T81) and (T83) have energies about 10 kcal mol'1 lower than those for the E2
elimination reactions (T85), (T86), (T87) and (T88). Thus, given that the significant
difference in transition state barriers between the neighboring group and E2 elimination
reaction pathways suggest that pathways 1 and 2 are likely to be more favorable, it is
expected that the loss of CH38R from the side chain of the methionine fixed charge
sulfonium ion peptides will result in the formation of the five membered hydrofuran

and/or six membered oxazine product ions.

3.3 Potential Ring Opening of the Five and Six Membered Cyclic Product Ions via
Intramolecular Proton Transfer Reactions.

Having established above that the initial loss of CH3SR from the side chain fixed
charge sulfonium ion containing peptides following MS/MS is likely to occur via
neighboring group participation mechanisms involving pathways 1 or 2 in Scheme 3.1, it
was then considered whether the resultant cyclic product ions could undergo ring opening
to yield the acyclic vinyl glycine containing product, via intramolecular proton transfer
reactions according to pathways 1' and 2' in Scheme 3.1, prior to their further dissociation
by M33.

Structure (T82) in Figure 3.1(A) shows the predicted low energy transition state for the
ring opening reaction of the five membered hydrofuran product ion via pathway 1’ in

Scheme 3.1. The activation barrier for pathway 1' was predicted to be 45.0 kcal mol'1

47

 
 
    

1.86A 2.33A 1.86A 241A
A Pathway 3 -2

1.361 1.4011 1Q;
. 1.29A Pa‘hwaY“ 125A.\
2.12A
1.87A 82.27A

(T57)
-.(24kcal mol' 28.7 kcalmol‘l 4.9kc§l)'mol' 28.3(1tcalmolI

l l

2.64 A
+ S(CH3)2 ‘— +S(CH3)2‘_
9»
1.76 A ‘ fl 2.01 A

2.47

217A

1.73 A "86 A
(Q) (P) (T) (S)
14.4 kcal mol‘l 7.3 kcal mol'l 16.0 kcal mol'| 9.4 kcal mol'I

Figure 3.3 Optimized precursor, transition state and product ion structures (at the B3 LYP/
6-31+G** + ZPVE level of theory) for the loss of CH38CH3 fi'om the simple model
peptide N-acetyl methionine—N-methyl amide involving the alternate intramolecular
proton (Bz-H) transfer to (A) the C-terrninal carbonyl group (Pathway 3-2) or (B) the N-
terrninal carbonyl group (Pathway 4-2) to yield protonated vinyl glycine. The energies of

all molecules are given with respect to structure A in Figure 3.1.

48

with respect to the energy of structure (A). The structure of transition state (T82)
associated with pathway 1’ is similar to the structure of transition state (T86) associated
with pathway 4, and result in the formation of identical product ions (compare structure
(D) in Figure 3.1A and structure (N) in Figure 3.28). However, it can be seen from
Figures 3.1 and 3.2 that transition state (T82) is predicted to have a significantly higher
energy (45.0 kcal mol") compared to transition state (T86) (28.7 kcal mol"). Justification
for this difference may be obtained by consideration of the bonds being cleaved in each
case i.e., transition state (T82) involves cleavage of a strong carbon oxygen bond,
whereas transition state (T86) involves cleavage of a weak carbon sulfur bond.

The predicted transition state barrier for pathway 2’ (transition state (184) in
Figure 3.1), involving ring opening of the six membered oxazine was predicted to be 45.3
kcal mol'l with respect to the energy of structure (A), which is comparable to that
predicted for ring Opening of the five membered ring in Pathway 1’. Similar to that
discussed above, the structure of transition state (T84) is similar to the structure of
transition state (T85) associated with pathway 3, and again result in the formation of
identical product ions (compare structure (H) in Figure 3.1B and structure (K) in Figure
3.2A). However, the relative energy of transition state (T85) was predicted to be
significantly lower than that for transition state (T84).

The transition state barriers for ring opening of the five membered hydrofuran
product ion via intramolecular proton transfer pathways 1’-2 and 2’-2, involving the
alternate hydrogens to those shown in Figure 3.1 were also determined (structures (T 89)

and (T810) in Figure 3.4). The predicted barriers relative to structure (A) for these

49

A B
1.49A 1.19A 1.524 1.4oA1.21A
Pathway I‘-2 1.63 A 1119 A Pathway 2’2 [“156 A
2.59 A —" ——’
2.22 A
5 1.99 A
(U) (T59) (G) (T310)
3.6 kcal mol'l 50.5 kcal mol" 0.0 kcal mol'1 56.] kcal mol'l

l

1.33 A
2.25 A
2‘ 2.65 A
1.35 A 3.01 A
(V) (W)
8.2 kcal mol‘l 18.0 kcal mol"

Figure 3.4. Optimized precursor, transition state and product ion structures (at the
B3 LYP/ 6-31+G** + ZPVE level of theory) for ring opening of the product ions formed .
from Pathways 1 and 2, involving the alternate intramolecular proton (Bz-H) transfer to
either the N-terminal carbonyl group (Pathway 1’ in A) or the C-terminal carbonyl group
(Pathway 2’ in B) to yield protonated vinyl glycine product ions. The energies of all

molecules are given with respect to structure A.

50

pathways were found to be 50.5 and 56.1 kcal mol", respectively. Thus, each of the
transition state barriers predicted for pathways 1’, 1’-2, 2’ and 2’-2 are all significantly
higher in energy than those associated with pathways 1, 2, 3, and 4.

Recently, Paizs et. al. have predicted that the transition state barriers for amide
bond cleavage reactions range from 29 to 33 kcal mol", depending on the proton mobility
[52]. Thus, the predicted low energy transition states (T8 1) and (T83) for the neutral loss
of CH3SR via the neighboring group participation reactions discussed above are around
15 kcal mol'l lower than these predicted amide bond fragmentation barriers. Thus, it is
expected that fragmentation of the methionine fixed charge sulfonium ion containing
peptide precursors under the ‘slow heating’ collision induced dissociation methods
commonly employed for tandem mass spectrometry [53], would preferentially result in
formation of the five membered hydrofuran and srx membered oxazine product ions.
Furthermore, given that the barriers to amide bond cleavage are expected to be much
lower than the barriers predicted for the ring opening reactions of these initial cyclic
product ions, it is expected that these ring opening reactions are unlikely to occur, and
that further dissociation of the initial product ions would result in fragmentations along
the peptide backbone. A summary of the predicted activation barriers associated with the
various mechanistic pathways for dissociation of methionine side chain fixed charge

containing sulfonium ion peptides discussed above is given in Figure 3.5.

51

Intramolecular Proton Transfer
E (45 kcal mol“)

 

 

'

E2 elimination
(27 kcal mol")

   
  
 

Neighboring Group
(14.3 kcal mol")

Vinyl
glycine

Vinyl
glycine

I"
' \
I \
\
‘ .
9
0 ‘
V 7
I
l
l
I
D
I

Hydrofuran / Oxazine

 

 

Figure 3.5. Summary of the predicted low energy transition state barriers (at the
B3LYP/6-3l+G** +ZPVE level of theory) for the dissociation of methionine side chain

fixed charge containing sulfonium ion peptides.

52

3.4 Obtaining Experimental Evidence for the Mechanisms Responsible for the Loss
of CH3SR from Methionine Fixed Charge Sulfonium Ion Containing Peptides.

In order to obtain experimental evidence to support the results from the
theoretical calculations discussed above, the multistage tandem mass spectrometry
(MS/MS and M83) fragmentation reactions of a sulfonium ion derivatized
regioselectively deuterated methionine containing peptide, as well as those of a simpler
alanine containing model peptide, have been examined in a linear quadrupole ion trap. It
can be seen from Scheme 3.1 that loss of the methionine fixed charge containing side
chain via either an E2 elimination reaction would involve transfer of one of the B-methyl
hydrogens in the methionine side chain, resulting in ‘mobilization’ of this hydrogen along
the peptide backbone. This ‘mobilization’ would also occur if either of the B-methyl
hydrogens were involved in intramolecular proton transfer ring opening reactions of the
cyclic five or six membered hydrofuran or oxazine product ions initially formed via the
neighboring group participation reactions. In either case, facile migration or ‘scrambling’
of this hydrogen (proton) along the peptide backbone would be expected prior to or

during further dissociation of the side chain cleavage product ion by MS3 [54].

3.4.1 Regioselective Deuterium Labeling Experiments to Examine Mechanisms for
the Side Chain Fragmentation Reactions of Methionine Fixed Charge Containing
Peptide Ions.

To determine whether or not facile scrambling of the methionine side chain [3-
methyl hydrogens does occur prior to or during M83 dissociation, the MSIMS and M83

fragmentation reactions of the synthetic peptide GAH.(d4-M)GAILK, containing a

53

methionine side chain fixed charged phenacylsulfonium ion derivative, and where the
hydrogens at the B and 7 carbon positions of the methionine side chain were
re gioselectively exchanged for deuterium, were examined.

Figures 3.6A and B show the CID MSIMS product ion spectra of the doubly
[M’i-t-H]2+ and triply [M++2H]3+ charged precursor ions of GAIL(d4-M(R)GAILK, where
R = CH2COC6H5. From the doubly charged precursor ion product spectrum in Figure
3.6A, exclusive neutral loss of the fixed charge methionine side chain CH3SR was
observed. By examination of the product ion spectra obtained from the triply charged
precursor in Figure 3.6B, it can be seen that the neutral loss of CH3SR is also
accompanied by ‘charged’ loss of the fixed charge methionine side chain, giving rise to
complementary —(CH3SR+H+) and CH38R+H+ product ions. The formation of these
protonated ‘charged loss’ product ions indicate that the transfer of a deuterium atom via
an intermolecular deuterium transfer from the B methyl group of the methionine side
chain similar to pathways 1’ or 2’ in Scheme 3.1 (see Scheme 3.2) does not occur.
Therefore, this result provides evidence to support the contention that fragmentation of
the fixed charge methionine side chain does not result in the formation of a vinyl glycine
containing product ion.

Figure 3.6C shows the MS3 product ion spectrum obtained by dissociation of
the neutral loss [M++H-CH3SR]2+ product ion of the [M++H]2+ precursor of GAIL(d4-
M)GAILK from Figure 3.6A, from which a clear series of b- and y-type ions are
observed. As mentioned above, if dissociation of the fixed charge containing precursor
ion occurs via an E2 elimination reaction, or if ring opening of the initial cyclic product

ion occurs via intramolecular proton transfer, a deuterium atom from the [3 methyl group

54

 

 

 

 

 

 

 

 

 

 

 

 

100 ‘
A
_. [M++H]2+
1 l
2110i 4110’ .600197807011'1000 11200
8 100 _ -CH3SR
g _ B
“E 3 -(CH,SR+H+)
q, ‘ [M++2H]3*
.g ~CH3SR+Ht l
.‘3 l 1 .. ---
q) . 1 . . r 3 . . r 4 ~ . 4 4 w . r . 1%
CZ 200 400 600 800 1000 1200
_ Y5
100
2
3 C yr;+ 87Da
‘——>
r 87Da Y6 Y7 b9
3 F‘B b
3 y] b3 :94 5 b7 8 Y8
l 1;-121 12JL1111-111 .11- -11-- - T11 I
200 400 600 800 1000
m/z

Figure 3.6 Linear ion trap CID MSn analysis of a methionine side chain phenacyl

sulfonium ion fixed charge derivative of GAIL(d4-M)GAILK. (A) CID MSIMS product

ion spectrum of the [M++H]2+ ion. (B) CID MSIMS product ion spectrum of the

[M"+2H]3+ ion. (C) CID M33 product ion spectrum of [M++H-CH3SR]2+ ion from Figure

3.6A.

55

"CH3s

D
‘1’ f *
+ CH SR+D
1 9" $6 /“ 3

 

 

c N c
/ \ c
Pathway] E H}? H H111\
\
\+/R
5)
Pathway 2 Pathway 1
‘11) \
/C<1§1 céO
H
1.11
r _
D 01,516 D
Pathwa 2 P o
y 9») D II 1 + CH3SR+D+
/C<;i *0 /C\N c¢O
H 1 H I
HN\ "N\

 

 

Scheme 3.2. Potential intermolecular deuterium transfer reactions for the charged loss of

CH38R+D+ from methionine side chain fixed charge containing peptide ions.

56

of the methionine side chain would be mobilized in the resultant product ion to a position
along the peptide backbone. As a result, statistical ‘scrambling’ of this deuterium along
the peptide backbone would be expected. However, it can be seen from Figure 3.6C that a
mass difference of 87 Da is observed between the ys and y6 product ions, as well as
between the b4 and b5 product ions. These ions represent product ions formed by
fragmentation of the amide bonds on the N and C-terminal sides of the methionine
residue. This mass difference, as well as the lack of evidence for deuterium incorporation
into any of the other product ions formed, indicates that all four deuterium atoms are
located in the side chain of the product ion, and are therefore not involved in the initial
fragmentation reaction. This data further indicates that the E2 elimination and ring
opening reactions are not involved in fragmentation of the fixed charged methionine side
chain sulfonium ion peptides, and is therefore strongly suggestive of the formation of

cyclic hydrofuran and oxazine product ions.

3.4.2 Multistage Tandem Mass Spectrometry Experiments to Examine the
Structure(s) of the Product Ions formed by Side Chain Fragmentation of
Methionine Fixed Charge Containing Peptides.

The MSIMS and MS3 data obtained from the deuterium labeling experiments
above indicate that fragmentation of methionine side chain fixed charge sulfonium ion
containing peptides results in the formation of cyclic five and/or six membered
hydrofuran or oxazine containing product ions via neighboring group participation
reaction mechanisms, and that these product ions do not undergo ring opening via

intramolecular proton transfer reactions prior to further dissociation.

57

Scheme 3.3 shows the proposed structures of the five and six membered product ion
formed via the loss of CH3SR from GAILM(R)GAILK (Structures A and B, respectively)
and the structure of a ‘control’ peptide GAILAGAILK (structure C). The fragmentation
of product ions containing these cyclic structures (e.g., the y6 and y7 ions from structure
A, and the y7 ion from structure B) should differ from the fragmentation of the same
product ions formed from a ‘control’ peptide GAILAGAILK lacking these cyclic
structures (structure C in Scheme 3.3). Therefore, in order to examine the structure(s) of
the initial product ions formed by side chain fragmentation of methionine fixed charge
containing peptides, the multistage (MS3 and M84) product ion spectra of the doubly
charged neutral loss product ion from the GAILM(R)GAILK peptide, as well as the
MSIMS and M83 product ion spectra of the doubly charged ‘control’ peptide
GAILAGAILK peptide have been obtained.

Figures 3.7A shows the mass spectra obtained by MS3 of the neutral loss
[M++H-CH3SR]2+ product ion from the doubly charged precursor of GAILM(R)GAILK,
while Figures 3.7B and 3.7C show the mass spectra obtained by MS4 of the y6 and y7
product ions from Figure 3.7A, respectively. Figures 3.7D-F show the MS/MS spectrum
obtained from the [M+2H]2+ precursor ion, as well as the MS3 spectra obtained from the
resultant yo and y7 ions. Clearly, the spectra obtained by dissociation of the [W+H-
CH3SR12+ ion from GAILM(R)GAILK and the [M+2H]2+ ion of GAILAGAILK are quite
similar, with a complete series of b- and y-type ions observed in each case. Although y5
and y6 product ions corresponding to fragmentation on either side of the methionine side
chain in the GAH.M(R)GAILK peptide are both observed, fragmentation to yield the y5

ion is unlikely for the cyclic five membered hydrofuran containing product ion,

58

o o o
H II
GAl—C N—CH-ICI C/—r-N—CH2-C—A1LK

CH2 :y

”3

o o o 0
|| || || 11 ll
GAi—C N—CH-C CH— C N—CHz-C—AILK

y7CH-CH3 y6

”3

Scheme 3.3 Structures of (A) the five membered hydrofuran product ion formed via the
loss of CH3SR from GAILM(R)GAILK, (B) the six membered oxazine product ion
formed via the loss of CH3SR from GAILM(R)GAILK, and (C) GAILAGAILK. The
amide bond cleavage sites for formation of the y5, yr, and y7 product ions in each case are

indicated.

59

 

 

 

 

 

 

 

 

 

 

 

 

 

100~ ys
A
‘ yfi’ 83Da
1H
y‘ Y1 b9
b5
b3 b4 b7 b8 Y8
yl
. -l .. - l.
200 400 600 800 1000
100- NH3
6 B
E
s:
:1
.o
<
0 -1
.2
E b,+HZO
” b+HO '
a: b,+H,o b 4 2 b3!
11 1412 1 1 .1 I L
250 350 450 550 650
i
‘ -H O
4 \NH,
1‘ 1‘ L J_. . 1 Lry?- I
200 300 400 500 600 700 800
m/z

 

 

 

 

 

 

 

1’.-l. _‘ .-lyl

 

 

 

600'800‘10'00

y. r b5+HZO
b, or y:
Y3 . 1 ¥6

 

—4

 

AAA- 1

a Y
b 4
3.111. 21.1 .

250 350

 

 

 

 

‘ r

 

450 550 650

b6

b or °
1364-1120 71 Y7
1’1

 

11 [611,6 b

 

200

300 400

500 600 700 800
m/z

Figure 3.7 Multistage (MS/MS, MS3 and M84) tandem mass spectrometry analysis of

GAILM(R)GAILK (where R=CH2COC6H5) and GAILAGAILK. (A) CID Ms3 product

ion spectrum of the [M++H-CH3SR]2+ ion from GAILM(R)GAILK. (B) CID Ms4

product ion spectrum of the y5 product ion from Figure 3.7A. (C) CID MS4 product ion

spectrum of the y7 product ion fi'om Figure 3.7A. (D) CID MS/MS product ion spectrum

of the [M+2H]2+ ion of GAILAGAILK (E) CID MS3 product ion spectrum of the y6

product ion from Figure 3.7D. (F) CID MS3 product ion spectrum of the y7 product ion

from Figure 3.7D.

60

due to the double bond character of the amide bond, whereas fragmentation to yield the
y6 ion is not likely to occur in the cyclic six membered oxazine containing product ion
due to the presence of the oxazine ring. Thus, the fact that both ions are observed in
Figure 3.7A suggests that both cyclic products may be formed, consistent with the results
from the theoretical transition state calculations described earlier.

It can be seen by comparison of Figure 3.7B with 3.7E and Figure 3.7C with
3.7F that the product ion spectra obtained by dissociation of the y6 and y7 ions from the
two peptides are very different. It is expected that these differences are due to the
presence of the cyclic structures in the GAILM(R)GAILK peptide upon loss of the side
chain. The series of product ions observed in Figure 3.7B and F by dissociation of the y6
and y7 ions from the [M+2H]2+ ion of the GAILAGAILK peptide correspond to a
relatively complete series of a-, b-, and y-type ions. In contrast, the yr, ion from the
[M"+H-CH3SR]2+ ion of the GAILM(R)GAILK peptide may contain only the five
membered hydrofuran ring, whereas the y7 product ion may contain either the five
membered hydrofuran or six membered oxazine rings. The dominant product ions
observed by dissociation of these yrs and y7 ions (Figure 3.7B and 3.7C, respectively)
correspond to the loss of NH3 and/or the formation of a yr, ion. The loss of N H3 in Figures
3.7B is consistent with fragmentation of the five membered hydrofuran ring containing y6
precursor ion, while the formation of the y5 ion in Figure 3.7C is consistent with

fragmentation of the six membered oxazine ring containing y7 product ion.

61

3.5 Conclusion

Taken together, the results from the theoretical calculations and experimental data
presented here indicate that the loss of CH3SR from methionine side chain fixed charge
sulfonium ion containing peptide ions occurs via neighboring group participation
reactions, resulting in the formation of a mixture of both five membered hydrofuran and
six membered oxazine product ions. The data also suggests that these cyclic product ions
do not rearrange by intermolecular proton transfer reactions prior to or during further
dissociation. However, the resultant product ion spectra obtained by MS3 are not
significantly different from the spectra obtained from ‘control’ acyclic peptides,
particularly with respect to the formation of product ions resulting from cleavage of
amide bonds either side of the site of the loss of the fixed charge, and is likely due to the

presence of a mixture of both cyclic structures formed from the initial neutral loss.

62

CHAPTER FOUR
Substituent Effects on the Fragmentation Reactions of Methionine Side Chain Fixed

Charge Phenacylsulfonium Ion Containing Peptides.

4.1 Introduction

It was observed in Chapter 3 that fragmentation of the triply charged
phenacylsulfonium ion derivative of GAILM(R)GAILK resulted in the formation of
protonated ‘charged loss’ product ions in addition to the expected neutral loss product
(Figure 3.6 B). These charged loss products are formed via intermolecular proton transfer
from the triply protonated peptide product ion to the methylphenacylsulfide side chain
fragment following the initial fragmentation reaction. Control over the formation of the
characteristic low m/z charged loss product ion could potentially be used to selectively
identify the presence of fixed charge methionine containing peptides from within
complex mixtures by using a precursor ion scan mode MSIMS experiment. Alternatively,
the complementary high m/z charged loss peptide product ion could be used for selective
identification of the fixed charge methionine containing peptides by using a ‘neutral gain’
MSIMS scan [55]. It is expected that the ability for proton transfer from the peptide
product ion to the side chain fragment will vary according to differences in their
respective proton affinities. The proton affinity of the neutral side chain fragment may
potentially be modulated as a function of the identity of the para-substituent of the
alkylating reagent employed for the initial fixed charge derivatization. Substituents that
result in the formation of a more acidic neutral molecule should result in more abundant

neutral loss, while more basic substituents should result in more abundant charged losses.

63

However, charged losses are only expected when the proton affinity of the resultant
peptide product ion is relatively low i.e., when the number of total charges on the
resultant peptide product ion exceeds that of the number of basic sites within the ion.
Here, the theoretical proton affinities of a series of para-substituted (-COOH (structure 6
in Figure 4.1), -COOCH3 (structure 7 in Figure 4.1), -H (structure 8 in Figure 4.1), -CH3
(structure 9 in Figure 4.1) and -CH2CH3 (structure 10 in Figure 4.1)) side chain fragments
have been calculated by using high level density functional theory calculations of the
neutral species and protonated ions at the B3LYP/6-3l+G** +ZPVE level of theory.
Then, the influence of these side chain fragment proton affinities on the relative
abundance of neutral versus charged side chain losses resulting from the dissociation of
methionine fixed charge containing peptide ions have been determined by examination of
the fragmentation reactions of a series of para-substituted phenacylsulfonium ion

containing peptides.

4.2 Proton Affinity Calculations

The structures of the various para-substituted neutral side chain fragments formed
via cleavage of the side chain methionine fixed charge sulfonium ion peptides, as well as
their protonated ions, were initially subjected to full conformational searches at the PM3
level of theory. Then, low energy conformers were re-optimized at the B3LYP/6-31+G**
density functional level. Vibrational frequency analysis was then performed to determine
the nature of the optimized stationary point structure, and to obtain values for the zero
point vibrational energies (ZPVE) for subsequent proton affinity calculations. Figure 4.1

shows the optimized low energy structures for each of the neutral species and protonated

ions determined from these calculationsnThe proton affinities of the neutral structures
were then calculated according to the method described in section 2.9.3 of Chapter 2. The
proton affinities derived from these calculations are given in Table 4.1. According to
these results, the proton affinity of the neutral fragment increases from 210.6 — 219.0 kcal
mol'1 in the order of structures 6, 7, 8, 9 and 10. These results are consistent as being due
to the expected changes in the electron density of the fragments, as determined from the

Hammett equation o-values for the substituents (Table 4.1) [56,57,58].

4.3 Determining the Influence of Side Chain Fragment Proton Affinities on the
Relative Abundance of Neutral versus Charged Side Chain Losses from Methionine
Fixed Charge Containing Peptide Ions.

Figure 4.2A-C shows the CID MSIMS product ion spectra obtained by
dissociation of the singly [M]", doubly [M++H]2+ and triply [M++2H]3+ charged
methionine side chain fixed charge phenacylsulfonium ion derivative of the synthetic
peptide GAILMGAILK, respectively. It can be seen from these figures that dissociation
of the singly and doubly charged precursor ions results in exclusive neutral loss of the
side chain (labeled as —CH3SR). In contrast, dissociation of the triply charged precursor
ion results in the formation of both neutral loss (—CH3SR) and charged loss (labeled as -
(CH3SR+H+) and CH38R+H+) product ions. This result is consistent with the expected
proton affinities of the resultant peptide product ions. The proton affinity of the peptide
product is expected to decrease with increasing charge state. The ionizing proton on the
singly charged peptide product ion is likely to reside at either the 8-amino group of the

lysine side chain or along the peptide backbone, while the two ionizing protons on the

65

11

 

. A
209 I
7 12
2.70:5v 2.10.11,
8
2.72‘A/

 

   

2.72 A; 2.11 A

Figure 4.1 Optimized low energy structures at the B3LYP/ 6-31+G** + ZPVE level of
theory for the neutral and protonated products of the para-substituted

methylphenacylsulfide derivatives employed for proton affinity calculations.

66

Table 4.1 Total energies (Emmi), zero point vibrational energies (ZPVE), vibrational

energies (Evrbmgmr) and proton affinities computed at the B3LYP/6—31+G** level of

theory for the para-substituted methylphenacylsulfide derivatives.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

P 31’ 3' S 1111C ture EToml ZPVE EVibrational PI 0101] Affinity
substituent (Hartree) (kcal mol") (kcal mol") (kcal mol'l)
_ C OOH Neutral (6) -101 1.009419 1 14.375630 121 .249000 210.6
”08‘3“" -101 1.354606 121.810810 128.692000
_ C o 0013 Neutral (7) -1050.3l664 131.888080 139.810000 212.8
”0:83?“ -1050.662343 139.323310 147.298000
_H Neutral (8) -822.424349 105.043650 1 10.1 19000 215.0
“0(1):?th -822.776703 112.701460 1 17.755000
CH Neutral (9) -861 .746192 122.103150 128.38900 218.5
" 3
Prom?“ -862. 104304 129.788560 136.04300
-CH2CH3 Neutral (10) -901 .063164 140.288790 147.256000 219.0
”08;?“ -901422005 147.935160 154.93900

 

67

 

-CH3SR

 

 

 

 

 

 

 

 

 

 

 

 

100' A 938.3
fl
M+
‘ 1T5
400 600 800 1000 1200 1400
o 100 -CH3SR
8 l B 469.6
CG a
“U
G
3 _
< [M++H]2+
a) 7 522.5
.2
. ~ 1
2
go 200 400 600 800 1000 1200 1400
-CH3SR
100 ‘ 313.4
‘ [M++2H]3+
‘ 368‘s -(CH3SR+H+)
_ 469.7
CH3SR+H+
‘167.0
1 1 1 -
200 400 600 800 1000 1200 1400

m/z

Figure 4.2 Linear ion trap CID MS/MS analysis of methionine side chain fixed charge
phenacylsulfonium ions of GAILMGAILK. (A) CID MSIMS product ion spectrum of the
[M+] ion. (B) CID MSIMS product ion spectrum of the [M“+H]+2 ion. (C) CID MS/MS

product ion spectrum of the [M++2H]+3 ion.

68

doubly charged peptide product ion are likely to reside at both side chain and backbone
positions. In the case of the triply charged peptide product ion however, increased
columbic repulsion, as well as a decrease in the local proton affinities of the remaining
sites potentially available for protonation, are likely to result in intermolecular proton
transfer from the peptide fragment to the side chain fragment following bond cleavage,
but prior to separation of the initial ion-neutral complex.

The fragmentation reactions of the multiply charged precursor ions of the
methionine side chain fixed charge phenacylsulfonium ion containing synthetic peptides
VTMGHFDNFGR and VTMAHFWNFGK were also examined (Figure 4.3 and Figure
4.4, respectively). Similar to that discussed above for dissociation of the GAILMGAILK
peptide ions, dissociation of VTMGHFDNFGR and VTMAHFWNFGK resulted in the
exclusive neutral loss of the side chain from the low chare state precursor ions ([M"+H]2+
and [M++3H]3+), where the ionizing protons in the resultant peptide product ions could be
accommodated at the side chains of the lysine, arginine and histidine amino acids within
the sequence or along the peptide backbone. For the quadruply charged ([M++4H]4+)
precursor ions however, extensive charged losses are observed. Interestingly, when the
CID MS/MS product ion spectra of the triply charged GAILMGAILK, and the quadruply
charged VTMGHFDNFGR and VTMAHFWNFGK peptide ions are examined, it can be
seen that the abundance of the neutral versus charged loss product ions formed are highly
dependant on the charge state and amino acid sequence of the peptide precursor. The

relative abundance of neutral versus charged loss product ions observed from the fixed

charge containing peptides is expected to vary according to the differences in proton

69

 

-CH3SR

 

 

 

 

 

 

 

 

 

 

 

 

100- 616.9
A
_ [M"+H]2+
6919.8
200 400 600 800 1000 1200 1400
v 100 a CH SR
é B 4113.5
1, _
C.‘
= .
..D
< - [M++2H]3+
é" 416.8
‘5 _
'75 ..- l a .-
m AI I I T I I I
§ 200 400 600 800 1000 1200 1400
-(CH3SR+H+)
1001 C 411.5
. -CH3SR
308.9
_ [M++3H]4+
- CH3SR+H+ / 350-6
167.0 (
l l Jm 1. l -a . m
200 400 600 800 1000 1200 1400
m/z

Figure 4.3 Linear ion trap CID MSIMS analysis of methionine side chain fixed charge

phenacylsulfonium ions of VTMGHFDNFGR. (A) CID MSIMS product ion spectrum of

the [M‘“+H]2+ ion. (B) CID MSIMS product ion spectrum of the [M++2H]3+ ion. (C) CID

MSIMS product ion spectrum of the [M++3H]4+ ion.

70

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 -CH3SR
100 A 645.4
4
- [M++H]2+
728.1
200 400 600 800 1000 1200 1400
a) _ -CH SR
8100 B 430.35
5; .
c:
3 -
< [M++2H]3+
E J 485.7
‘5 * 1
be 200 400 600 800 1000 1200 1400
-(CH3SR+H+)
I -CH3SR
- 323.2 [M++3H]4+
364.6
. CH3SR+H+ /
167.0 f
200 400 600 800 1000 1200 1400
m/z

Figure 4.4 Linear ion trap CID MSIMS analysis of methionine side chain fixed charge

phenacylsulfonium ions of VTMAHFWNFGK. (A) CID MSIMS product ion spectrum of

the [M“+H]2+ ion. (B) CID MSIMS product ion spectrum of the [MW-2H]3+ ion. (C) CID

MSIMS product ion spectrum of the [MW-3H]4+ ion.

71

affinity between the peptide product ions and the neutral side chain fragment. Thus, from
the results observed above, it can be determined that the proton affinities of the triply
charged VTMGHFDNFGR and VTMAHFWNFGK product ions are both lower than that
of the doubly charged GAILMGAILK product ion, and that the proton affinity of the
triply charged VTMAHFWNFGK product ion is lower than that of the triply charged
VTMGHFDNFGR product ion.

Previous studies carried out to examine the effect of proton affinity on the ratios
of product ion abundances formed by CID have observed a linear relationship between
the log of the ratio of the b2 and yr type product ion abundances and the proton affinities
of the C-terminal amino acid for a series of protonated tripeptides Gly-Gly-Xxx (where
Xxx represents various amino acid residues) [59]. Here, to determine the effect of the
leaving group proton affinities on the ratio of neutral versus charged loss from the
methionine side chain fixed charge sulfonium ion containing peptides, the fragmentation
of a series of para-substituted phenacylsulfonium ion containing peptides have been
examined. Figures 4.5A-D show the CID MSIMS product ion spectra obtained from the
triply charged [M+2H“]3+ precursor ions of GAILMGAILK derivatized at the methionine
side chains using the para-benzoic acid (Figure 4.5A), para-methylbenzoate (Figure
4.5A), para-methyl (Figure 4.5A) and para-ethyl (Figure 4.5A) substituted
phenacylbromide reagents (Compounds 2-5 in Chapter 2, respectively). Figures 4.6 and
4.7 show the CID MSIMS spectra obtained by dissociation of the quadruply charged
[M+3H+]4+ precursor ions of VTMGHFDNFGR and VTMAHFWNFGK, respectively,
each formed by derivatization at their methionine side chains using the same reagents as

above.

72

-CH3SR 100 -CH3SR

 

 

 

00
8 313.4 A 313.5 B
E
‘2 CH3SR+H*
5 3 225.1

M+ 211*
9 583.1 ' IM*+2HI3*
E .CH35R+H* 38’“
§ 469.6 .CH35R+H*
Q / 469.8
a

l I ‘ v V I L L1

200 400 600 800 l000 1200 I400 200 400 600 800 1000 1200 1400
-CH,SR+H' -(CH,SR+H‘)

U ‘00 469.7 C 100 D
U
G
CB
'0
S

+
g 3133“" CH3SR+Ht
u ‘ [M"+2Hl3* 195.0
.3 .cthsg 373.5 [M++2H]3*
T“: 313.5 £11,511 3789
a: 3135
e I | l

200 400 600 800 1000 l200 I400 200 400 600 800 1000 1200 1400
m/z m/z

Figure 4.5 Linear ion trap CID MS/MS analysis of methionine side chain fixed charge
para-substituted phenacylsulfonium ions of GAILMGAILK. (A) CID MSIMS product
ion spectrum of the para-benzoic acid phenacylsulfonium [M++2H]3+ ion. (B) CID
MSIMS product ion spectrum of the para-methylbenzoate phenacylsulfonium [M++2H]3"
ion. (C) CID MSIMS product ion spectrum of the para-methyl phenacylsulfonium
[M++2H]3+ ion, and (D) CID MSIMS product ion spectrum of the para-ethyl

phenacylsulfonium [M++2H]3+ ion.

73

 

-CH3SR

 

 

 

100- 308.8
g, A
5.; -CH3SR+H+
a 411.5
g 5333“”
.3 // I
3 ( [M*+3Hl4+
0
a: . / 361.6
f M
200 400 600 800 1000 1200 1400
‘ ‘CH38R+H’
@100 411.5 C
U
5
: c113sa+ll+
.‘3
.o - 181.0
<
23 303-8 [M*+3H]“*
2'5 353.8
a: 4 /
e 1 (1!

 

 

 

200 400 600 800 10001200 1400
m/z

1003

100‘

CH 3SR+H*
1 225\.0 /

-CH3SR+H*
41 1.5

-CH3SR
/ 308.9

[M*+3H]“

 

 

.10:

200400600800

CH3SR+H+

‘ 195.0

~CH3SR

 

‘ 301.8

*——r_

‘CH3SR+H+
41 1.5

[M’+3H]‘“
/ 357.8

 

200400600800

mlz

J

1000 1200 1400

D

1000 1200 1400

Figure 4.6 Linear ion trap CID MSIMS analysis of methionine side chain fixed charge

paraesubstituted phenacylsulfonium ions of VTMGHFDNFGR. (A) CID MSIMS product

ion spectrum of the para-benzoic acid phenacylsulfonium [M++3H]4+ ion. (B) CID

MSIMS product ion spectrum of the para-methylbenzoate phenacylsulfonium [W+3H]

4+

ion. (C) CID MSIMS product ion spectrum of the para-methyl phenacylsulfonium

[M“+3H]4+ ion, and (D) CID MSIMS product ion spectrum of the para-ethyl

phenacylsulfonium [M++3H]4+ ion.

74

 

 

-CH3SR+H*

 

 

 

 

100-

8 430.6 A

C

63 4

'0

g c113512+11+

D 211.0

‘5 {113512

‘>_’ \ 323.1

g x [M++3H14+

a: / 375.6

e K

200 400 600 800100012001400

-CH3SR+H*

100‘ 430.5 C

8

G

g

g c113sa+w

.o .1810

<2

9; £11351:

33 323.2 [M*+3H]‘“

- 368.4

é ~ ’

a l

200 400 600 800 1000 1200 I400
m/z

100‘

‘ 225.0

 

CH3SR+H‘

-CH3SR
323.1

11/

 

-CH3SR+H*
430.5

[M‘+3H]‘*

/ 379.10

 

‘ CH3SR+H‘
‘ -c113sn
323.2

11hr

200400600

195.0

 

800
-CH,SR+H’
430.6

[M’+3H]4+

/ 37l.8

1200 1400

D

 

 

 

2004006008001000

m/z

[200 1400

Figure 4.7 Linear ion trap CID MSIMS analysis of methionine side chain fixed charge

para-substituted phenacylsulfonium ions of VTMAHFWNFGK. (A) CID MSIMS

product ion spectrum of the para-benzoic acid phenacylsulfonium [M"+3H]4+ ion. (B)

CID MSIMS product ion spectrum of the para-methylbenzoate phenacylsulfonium

[M++3H]4+ ion.

(C) CID MSIMS product ion spectrum of the para-methyl

phenacylsulfonium [MW-3H]4+ ion, and (D) CID MSIMS product ion spectrum of the

para-ethyl phenacylsulfonium [M++3H]4+ ion.

75

It can be seen from Figures 4.5, 4.6 and 4.7 that the ratio of neutral versus charged
losses from the methionine side chain of these precursor ions varies as a function of the
identity of the neutral side chain fragment, and that the extent of charged loss product
ions increases with increasing proton affinity of the neutral side chain fragment. For
example, when the triply charged para-benzoic acid substituted phenacylsulfonium ion
derivative of GAILMGAIK was subjected to CID-MSIMS (Figure 4.5A), the neutral loss
of the side chain was observed as the dominant fragmentation pathway. This result
indicates that the proton affinity of the neutral side chain fragment (calculated as 210.6
kcal mol", see structure 6 in Table 4.1) is lower than that of the doubly protonated
peptide product ion. In contrast, CID-MSIMS of the triply charged para-ethyl
phenacylsulfonium ion derivative of the same peptide gave rise to almost exclusive
charged loss of the side chain. Thus, in this case, the proton affinity of the neutral side
chain fragment (calculated as 219.0 kcal mol", see structure 10in Table 4.1) must have a
higher proton affinity compared to the doubly protonated peptide product ion. These data
therefore provide a means to ‘bracket’ the proton affinity of the doubly charged peptide
product ion to between these two values. Similar proton affinity bracketing of the triply
charged product ions of VTMGHFDNFGR and VTMAHFWNFGK was obtained by
dissociation of the quadruply charged substituted phenacylsulfonium ion derivatives.

The plots shown in Figure 4.8 show a linear correlation between the log of the
experimentally observed ratios of the charged versus neutral loss product ion abundances
and the theoretically calculated proton affinities for the para-subtituted
methylphenacylsulfide side chain fragments of GAILMGAILK (Figure 4.8A),

VTMGHFDNFGR (Figure 4.83) and VTMAHFWNFGK (Figure 4.8C).

76

41 A

 

   
 
 
 
 

A ‘\
+ \
=5
x
a C "\ \‘c
+ 0 \208 2r04._ 212 214 \246 218 220
i“ °\., 7“ Proton Affinity (kcal mol")
5 \., ~\
5) 2 . \..\ I ‘.‘o
f ' "\.. R2: 0.9988
8
\

'12., 'R2=0.9851
:1:
E.) -6‘
5 ‘R2=0.9605

-8‘

 

Figure 4.8. Plot of ln[-CH3SR / (-(CH3SR+H+) + CH3SR+H+)] versus calculated proton
affinity for the phenacylsulfide derivatives of (A) the [M+2H]2+ side chain cleavage
product ion of GAILMGAILK (B) the [M+3H]3+ side chain cleavage product ion of
VTMGHFDNFGR and (C) the [M+3H]3+ side chain cleavage product ion of

VTMAHFWNFGK.

77

In order to account for any charge state dependant detector response, and therefore to
obtain a closer approximation of the actual abundances of the neutral and charged loss
side chain cleavage product ions formed, the product ion abundances were first
normalized with respect to charge state (i.e., intensity divided by charge), assuming mass
detection response to be linear with respect to the charge state of the ion. The x-axis
intercept of these plots may be used to estimate the proton affinities of the doubly
charged product ion from GAILMGAILK (216 kcal mol"), and the triply charged
product ions of VTMGHFDNFGR (211 kcal mol'l) and VTMAHFWNFGK (207.5 kcal
mol'l). The proton affinity of the amide bonds along the peptide backbone may be
estimated from a simple model of the peptide amide bond, N-methyl acetarnide, which
has a proton affinity of 212.4 kcal mol'1 [60]. Thus, each of the values estimated from the
data above are consistent with the ionizing proton being located at a position along the

peptide backbone.

4.4 Conclusions and Future Directions

These data described above indicates that the ratio of neutral loss to charged loss
product ion abundances from fixed charge containing peptide ions varies according to the
differences between the proton affinities of the neutral side chain fragment and the
peptide product ion. Given that the ratio of charged versus neutral loss observed for a
given peptide is observed to change as a function of peptide ion charge state, specific
information regarding the number of basic sites contained within the peptide may
potentially be obtained by having control over this reaction. In the future, the potential for

utilizing this information, along with knowledge regarding the presence of a specific

78

amino acid residue (e.g., methionine), proteolytic enzyme cleavage specificity,
chromatographic retention time, or accurate peptide mass, will be examined for directly
identifying peptides from the sequence databases, without need for further structural
information. Several precedents supporting the concept of improving the specificity of
database searches for protein identification, by promoting the formation of specific
product ions through chemical derivatization e.g., enhanced C-terminal aspartic acid
cleavage from singly-charged peptides by conversion of lysine to homoarginine, or
enhanced N-terminal amino acid cleavage by phenylthiocarbamyl modification have

recently appeared in the literature [61].

79

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