THE INFLUENCE OF CATION AND “ALTERNATIVE” AMINO ACIDS ON THE FRAGMENTATION PATHWAYS OF METAL CATIONIZED AND PROTONATED PEPTIDES A Thesis by SILA OCHOLA B.ED, Egerton University, Nairobi , Kenya, 1998. Submitted to the Department of Chemistry and the faculty of the Graduate School of Wichita State University in partial fulfillment of the requirements for the degree of Master of Science MAY 2008 ©Copyright 2008 by Sila Oduor Ochola All Rights Reserved THE INFLUENCE OF CATION AND “ALTERNATIVE” AMINO ACIDS ON THE FRAGMENTATION PATHWAYS OF METAL CATIONIZED AND PROTONATED PEPTIDES I have examined the copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the Degree of Master of Science, with a major in Chemistry. Michael J. Van Stipdonk, Committee chair. We have read this thesis and recommend its acceptance. Erach R. Talaty, committee member. Francis D’Souza, committee member. Paul Rillema, committee member. William Parcell, committee member. iii ACKNOWLEDGEMENT I wish to express my sincere and heartfelt gratitude to my faculty advisor Dr. Michael J. VanStipdonk for his kindness and being able to guide me academically. I have benefited a lot in working in his research laboratory and my knowledge of peptide chemistry has really improved. I am very grateful to Dr. Erach Talaty for his guidance especially with the reaction mechanisms. He encouraged me to pursue my dreams such as applying for Ph.D studies in Chemistry. I do sincerely thank him for being a member of my committee. My sincere gratitude is with Dr. Francis D’souza for his guidance and being a great teacher. From him I have acquired a great deal of Analytical Chemistry knowledge and most of all I thank him for being a member of my committee. My sincere thanks to Dr. Paul Rillema, first for accepting to be a member of my committee. I do thank him for listening to my requests and for his teaching excellence. I have always benefited from his wise counsel. I am thankful to Dr William Parcell for kindly accepting to be in my committee and for his valuable input. I am very grateful to my research group members for their encouragement and constructive input in my thesis work. I wish to thank all the faculty and staff of Chemistry Department. The work described in this thesis was supported by a grant from the National Science Foundation (CAREER-0239800) and a Faculty Scholar Award from the Kansas Biomedical Research Infrastructure Network. Funds for the purchase of the ESIinstrumentation were provided by the Kansas NSF-EPSCoR Program and Wichita State University. iv ABSTRACT Tandem mass spectrometry and collision-induced dissociation (CID) are the “workhorse” methods for protein identification in proteomics investigations. Recent studies have demonstrated significant differences in the CID spectra of Li+, Na+ and silver cationized peptides, particularly with respect to the preferred product ions. For example, the former produce primarily (bn+17+Li)+ while the latter preferentially generates (bn-1+Ag)+ species. To improve our understanding of peptide fragmentation in general, three separate studies were initiated. The objective of the first study was to determine the CID patterns for thallium(I) cationized peptides and compare them to those from Ag, Na, and protonated analogues. The goal was to determine whether thallium, which represents a monovalent cation of relative hardness that differs from that of the group I metals, would demonstrate reaction pathways similar to group(I) cations or Ag(I). CID results show that the tendency to produce (bn+17+Tl)+ or (bn-1+Tl)+ depended significantly on the peptide sequence. Also, the multi-stage CID of Tl+ cationized peptides fails in the determination of the peptide sequence. The second objective of this research was to determine the influence of a 4aminomethylbenzoic acid (4AMBz) residue on the relative intensities of (b3-1+cat)+ and (b3+17+cat)+ fragment ions using tetrapeptides of the general formula A(4AMBz)AX and A(4AMBz)GX (where X = G, A, V). For Li+ and Na+ cationized versions of the peptides there was a significant increase in the intensity of (b3-1+cat)+ for the peptides that contain the 4AMBz residue, and in some cases the complete elimination of the (b3+17+cat)+ pathway. The influence of the 4AMBz residue is attributed to generation of a highly-conjugated oxazolinone species as (b3-1+cat)+, which increases the stability of this product relative to the rival (b3+17+cat)+ ion. This conclusion is supported by dissociation profiles, which suggest v that the energetic requirements for generation of (b3-1+cat)+ are significantly lower when the 4AMBz residue is positioned such that it should enhance formation of the conjugated oxazolinone. The objective of the third study was to determine the effect of the same residues on the formation of (b3-1+cat)+ products from metal (Li+, Na+ and Ag+) cationized peptides. The larger amino acids suppress formation of b3+ from protonated peptides with general sequence AAXG (where X=β-alanine, γ-aminobutyric acid or εaminocaproic acid), presumably due to the prohibitive effect of larger cyclic intermediates in the “oxazolone” pathway. However, abundant (b3-1+cat)+ products are generated from metal cationized versions of AAXG. Using a group of deuterium-labeled and exchanged peptides, we found that formation of (b3-1+cat)+ involves transfer of either amide or α-carbon position H atoms, and the tendency to transfer the atom from the α-carbon position increases with the size of the amino acid in position X. To account for the transfer of the H atom, a mechanism involving formation of a ketene product as (b3-1+cat)+ is proposed. vi TABLE OF CONTENTS CHAPTER PAGE CHAPTER I INTRODUCTION………………………………………………...1 CHAPTER II EXPERIMENTAL METHODS Mass Spectrometry……………………………………………….12 Electrospray Ionization…………………………………………..14 Quadrupole Ion trap Analyzer…………………………………...17 Tandem Mass Spectrometry……………………………………..20 Procedure………………………………………………………...24 CHAPTER III Collision Induced Dissociation of Tl+-cationized peptides……...28 CHAPTER IV Influence of a 4-aminomethylbenzoic acid residue on the competitive fragmentation pathways during CID of metal cationized peptides……………………………………………….48 CHAPTER V Formation of (b3-1+cat)+ Ions from Metal-cationized Tetrapeptides containing β-Alanine, γ-Aminobutyric Acid or ε-Aminocaproic Acid Residues……………………………………………………70 CHAPTER VI Conclusion……………………………………………………....97 REFERENCES ………………………………………………….........................100 vii LIST OF FIGURES FIGURE PAGE Figure 1.1 Biemann nomenclature for fragment ions bn and yn ions………………...3 Figure 1.2 Representative structures of protonated b and y ions……………………3 Figure 1.3 CID spectra of protonated AAXG……………………………………….11 Figure 2.1 The components of mass spectrometer………………………………….13 Figure 2.2 Principle of electrospray ionization……………………………………...16 Figure 2.3 Basic components of quadrupole ion trap analyzer……………………...19 Figure 2.4 Collision induced dissociation of precursor ions………………………...22 Figure 2.5 Principle of Tandem Mass Spectrometry………………………………...23 Figure 2.6 Multiple reaction vessel peptide synthesis apparatus……………………27 Figure 3.1 CID spectra of protonated and metal cationized VAAF…………………35 Figure 3.2 CID spectra of protonated and metal cationized YGGFL……………….36 Figure 3.3 CID spectra of protonated and metal cationized VGVAPG……………..37 Figure 3.4 Multi-stage CID of (LGGFL + Tl)+ product ions………………………..40 Figure 3.5 Multi-stage CID of (FGGLL + Tl)+ product ions………………………..41 Figure 3.6 Multi-stage CID of (FGGGL + Tl)+ product ions………………………..43 Figure 3.7 CID of (AGGFL + Tl)+ product ions…………………………………….44 Figure 4.1 Structures of the group of peptides analysed…………………………….62 Figure 4.2 CID spectra of protonated and metal cationized GGGA………………..63 Figure 4.3 CID spectra of protonated and metal cationized AAAG………………...64 Figure 4.4 CID spectra of protonated and metal cationized A(4AMBz)AG………...65 viii LIST OF FIGURES (CONTINUED) FIGURE PAGE Figure 4.5 CID spectra of Na+ cationized A(4AMBz)AX and A(4AMBz)GX……..66 Figure 4.6 CID spectra of Na+ cationized AA(4AMBz)G and (4AMBz)AAG……..67 Figure 4.7 CID profile of Na+ cationized AXGV (X= A or 4AMBz)……………….69 Figure 5.1 Sequence/structures of model peptides used to study the influence of βA, γAbu and Cap on formation of (b3-1+cat)+ from metal-cationized AAXG……………………………………………………………………86 Figure 5.2 CID (MS/MS) spectra generated from AA(γAbu)G series cationized by: (a) H+, (b) Li+, (c) Na+ and (d) 107Ag+ and 109Ag+……………………….87 Figure 5.3 Spectra generated by CID (MS/MS) of D+ cationized, fully deuteriumexchanged: (a) AAAG, (b) AA(βA)G, (c) AA(γAbu)G and (d) AA(Cap)G……………………………………………………………88 Figure 5.4 Spectra generated by CID (MS/MS) of Li+ cationized, fully deuteriumexchanged: (a) AAAG, (b) AA(βA)G, (c) AA(γAbu)G and (d)AA(Cap)G…………………………………………………………….89 Figure 5.5 Spectra generated by CID (MS/MS) of 109Ag+ cationized, fully deuteriumexchanged: (a) AAAG, (b) AA(βA)G, (c) AA(γAbu)G and (d) AA(Cap)G……………………………………………………………90 Figure 5.6 CID (MS/MS) spectra of Li+ cationized (a) AA(γAbu)G and (b)AA(α-d2-γAbu)G……………………………………………………..91 Figure 5.7 CID (MS/MS) spectra of Ag+ cationized (a) AA(γAbu)G and (b) AA(α-d2-γAbu)G…………………………………………………….92 Figure 5.8 CID (MS3) spectra of (a) (b3-1+Li)+Ag+ and (b) (b3-1+Ag)+ derived from AA(γAbu)G………………………………………………………………93 ix LIST OF SCHEMES SCHEME PAGE Scheme 1 Pathway to (b3)+ (1a) and (b3+17+cat)+ (1b) from AAAG through 5membered cyclic intermediates ….............................................................10 Scheme 2 Multi-stage CID scheme of thallium cationized LGGFL………………..40 Scheme 3 Multi-stage CID scheme of thallium cationized FGGLL………………..42 Scheme 4 Multi-stage CID scheme of thallium cationized FGGGL………………..42 Scheme 5 Multi-stage CID scheme of thallium cationized AGGFL………………..45 Scheme 6 Multi-stage CID scheme of sodium cationized AGGFL………………...45 Scheme 7 Multi-stage CID scheme of protonated AGGFL………………………...46 Scheme 8 Multi-stage CID scheme of silver cationized AGGFL…………………..47 Scheme 9 Reaction mechanism for formation of (b3)+ from A(4AMBz)AG …........68 Scheme 10 Pathway to (b3-1+cat)+ from metal cationized AA(α-d2-γAbu)G through 7membered cyclic intermediate, with transfer of amide position H atom...94 Scheme 11 Potential pathway to (b3-1+cat)+ from metal cationized AA(α-d2-γAbu)G through 7-membered cyclic intermediate, with transfer of α-carbon position H atom………………………………………………………….95 Scheme 12 Pathway to (b3-1+cat)+ from metal cationized AA(α-d2-γAbu)G through alternative ketene mechanism……………………………………………96 x LIST OF TABLES TABLE PAGE Table 1 Representative product ion distribution of protonated and metal cationized YGGFL, VGVAPG, WHWLQL peptides……………………………….38 Table 2 Representative product ion distributions of protonated and metal cationized GPA, FGG and GHG peptides……………………………….39 xi CHAPTER I Introduction Tandem mass spectrometry combined with soft ionization methods such as electrospray ionization and matrix assisted laser desorption ionization (MALDI) provides an effective approach for peptide characterization. The identity of a peptide can be determined by isolating cationized precursor ions using one mass spectrometry (MS) stage, ion-activation using methods such as collision induced dissociation (CID) or surface induced dissociation in a second MS stage, and then analysis of the fragmentation product ions using a third MS stage. The fragmentation or MS/MS spectrum is used to deduce the amino acid sequence of the original peptide. Protonated peptide may dissociate into a wide variety of fragment ions including the an, and bn ions which are the N-terminal fragments, and yn ions that are C-terminal fragments [1, 2] (figure 1.1). The reaction pathways leading to bn and yn ions have been explained qualitatively using the mobile “proton model” [3-16] and more generally by the “pathways in the competition” model [17]. The main tenet of the mobile proton model is that protons undergo intramolecular migration from the most basic group on a peptide to the site of cleavage, where they can weaken the C-N bond and make the carbonyl C atom more susceptible to intermolecular nucleophilic attack. The recently introduced pathways in competition fragmentation model provides a more general framework, taking into account additional features such as proton transfer and peptide isomerization, structures and transition state energies in the dissociation step, and the thermodynamics associated with the separation of ion-molecules into fragmentation products. 1 Based on several experimental and theoretical studies [18-26], it is now generally agreed upon that formation of b-and y ions involves the attack of the carbonyl carbon by the carbonyl oxygen of the amino acid on the N-terminal side, thus forming a fivemembered ring intermediate. C-N bond cleavage without proton transfer results in the b ion formation, while one additional proton transfer step leads to generation of a y ion. According to this mechanism b-ions have a cyclic structure whereas y ions are linear (figure 1.2). Because it is difficult to predict the type of fragment ions that will be formed for a given peptide, and identification of products that contain the N or C terminus is difficult without a priori knowledge of the sequence of the peptides, de novo sequencing of protonated peptides based on interpretation of fragmentation patterns alone has been a challenge. CID of alkali metal-cationized peptides has been studied as an alternative approach to peptide sequencing. There have been several studies reported in the literature on the CID of alkali metal-cationized peptides [27-37]. The dissociation of (M+cat)+ ions, where M=peptide and cat= alkali metal, is known to reveal the metal binding sites and also produce specific fragmentations that are different from those of (M+H)+ ions. The major difference between the CID of alkali metal-cationized peptides and protonated peptides is that the former predominantly form (bn+17+cat)+ while the latter forms bn and yn ions (figure 1.2). 2 b1 a1 H2N O CH b2 a2 C NH C CH CH NH b3 O NH C R O CH C OH R R x3 x2 y3 Figure 1.1 R O a3 x1 y2 y1 Biemann nomenclature for fragment ions where bn type ions contain N-terminus and yn type ions contain the C-terminus. R2 O O H2N N H R1 N H O R4 + + R3 H2N H OH O bn + Figure 1.2 O H N R5 yn+ representative structures of protonated b and y ions. The mechanism for formation of (bn+17+cat)+ involves the transfer of the hydroxyl group from the C-terminus of the peptide to the adjacent amino acid and subsequent loss of the residue mass of the C-terminal amino acid leading ultimately to the product of a new alkali cationized peptide lacking the original C-terminal residue (scheme1b). The rearrangement reaction can be particularly well suited for sequencing because the metal ion is retained in the N-terminal fragment ion, which can undergo the same fragmentation reaction multiple times in a multiple-stage CID experiment. Formation of (bn-1+cat)+ ion is proposed to take place via a nucleophilic attack on the carbonyl carbon at the cleavage site by the oxygen atom on the adjacent carbonyl group to the N-terminal side of the cleavage site( as shown for b3+ from AAAG in 3 scheme 1a). Several previous studies have shown remarkable differences between Ag+ and alkali metal cationized peptides [27, 35]. For example, the most prominent species observed during the multistage CID of alkali metal cationized leucine enkephalin are the (bn+17+cat)+ ions. At higher CID stages however, dissociation of the YG (b2+17+cat)+ results in the production of (a2-1+cat)+ species. In the investigation of YGGFL it was found that (b4-1+Ag)+ was favored over both the (b4+17+Ag)+ and (a4-1+Ag)+ ions at activation amplitude ranging from 20-30% of the excitation voltages accessible on the LCQ-DecaTM and an activation time of 30ms. The multiple-stage CID of Ag+ cationized leucine enkephalin can be initiated with either the (bn-1+Ag)+ or (bn+17+Ag)+ produced at the MS/MS stage. Multiple stage CID of (bn-1+Ag)+ proceeds through sequential elimination of CO to produce (an-1+Ag)+ ions and then subsequent loss of imine of the C-terminal amino acid to generate the next (b2-1+Ag)+ species. Similar to the alkali cationized peptides, CID of (b2-1+Ag)+ produces prominent (a2-1+Ag)+ ions. It has been noted that (b1) ions are not normally observed in the CID of protonated peptides and even with metal cationized peptides, formation of (b1-1+cat)+ is not the favored process. The lack of (b1-1+cat)+ is attributed to the fact that the mechanism for the generation of (bn) products involves the intervention of a five membered ring structure. This particular mechanism requires cleavage of a carbonyl moiety to the N-terminal side of the amide bond in the dissociation reaction. In the case of the (b2-1+cat)+ or (b2+17+cat)+ species, such a carbonyl group is not present and CID of these precursor ions leads primarily to the production of the cationized imines (a2-1+cat)+. 4 Recently, studies have shown that CID of Ag+-cationized peptides is more informative in terms of peptide sequencing than either alkali metal cationized peptides or protonated peptides [37]. Research studies by Siu and co-workers have demonstrated that the CID of argentinated peptides results in the production of (bn-1+cat)+ and (an-1+cat)+ ions which are similar to the CID of protonated peptides, while the production of (bn+17+cat)+ ions are in line with the CID of alkali metal cationized peptides [38]. The group of Siu and co-workers have found that the specific advantage of studying the CID of argentinated peptides is the fact that a prominent triplet of product ions consisting of (an-1+cat)+, (bn-1+cat)+ and (bn+17+cat)+ ions is consistently observed in the CID spectrum. This observation leads to direct determination of C-terminal amino acid. Despite nearly two decades of effort to resolve the mechanisms behind peptide dissociation, many details remain unclear. For this reason, three specific and focused studies were designed and carried out. These independent studies are related in that they are all designed to provide new information, or analytical approaches to generate new information, about the dissociation of gas-phase protonated and metal-cationized peptides. Results from these studies demonstrate that there are significant differences in the CID spectra of Ag+ and alkali (Li+ and Na+) cationized peptides. The former primarily form (bn-1+cat)+ and the later forms (bn+17+cat)+ as the major product ions respectively. For identical peptides, the product ions of (M+Ag)+ are more intense than those of (M+Na)+, and relative intensities of the (bn+OH+Ag)+ ions are typically lower than those of the (bn+OH+Na)+ ions. While the differences may be due to differences in hardness of the cations, testing of this hypothesis is difficult because of the lack of monovalent 5 transition metal ions to compare to Ag+. The objective of the first study was to investigate the CID patterns of Tl+ cationized peptide, and in particular whether thallium would demonstrate reaction pathway similar to group(I) cations or Ag+. The first set of experiments involved CID of protonated and metal cationized peptides followed by the multi-stage CID of the major product ions. Apart from the identity of cation (metal or hydrogen), the C-terminal amino acid can influence the fragmentation pathway. This phenomenon was illustrated by the earlier studies by Glish and co-workers who investigated the influence of C-terminal residue in the formation and abundance of (bn+17+cat)+ ions using a quadrupole ion trap mass spectrometer [34]. After acquiring a large number of MS/MS spectra of alkali cationized peptides, two general categories of dissociation behavior were determined based on which of the 20 common amino acids were at the C-terminus. Fifteen amino acids are in category I and five in category II. Peptides with C-terminated category I amino acids were found to form primarily the (bn-1+Na +OH)+ product ions. The category I amino acids include alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, lysine, histidine, cystine, arginine, glutamic acid, glycine and aspartic acid. Although peptides with C-terminated category II amino acids (tryptophan, serine, threonine, asparagine and glutamine) generally do not produce (bn-1+Na+OH)+ ion as the most abundant ion following CID, they do provide other alkali cationized analogs, such as bn-1 and characteristic fragments. These dissociation patterns combined with the capability of ion trapping instruments to induce multiple CID stages, provide opportunities for a new method in peptide sequencing. 6 In an attempt to enhance the understanding of how cation and sequence influence the peptide fragmentation, our group recently investigated the dissociation of metal cationized, model N-acetylated tetrapeptides, with the general sequence AcFGGX, that featured C-termini designed to allow transfer of the –OH required to generate the (b3+17+cat)+ product ion, but not necessarily as the most favored pathway [29]. The amino acid placed at position X either required a larger cylclic intermediate than the fivemembered ring presumably formed with α-amino acids (β-alanine, γ-aminobutyric acid and є-amino-n-caproic acid to generate six-, seven- or nine-membered rings, respectively) or prohibited cyclization because of the inclusion of a rigid ring (para- and meta-aminobenzoic acid). For Ag+, Li+, and Na+- cationized AcFGGX, formation of (b3+17+cat)+ was suppressed when the amino acids requiring adoption of larger ring intermediates were used, while amino acids that prohibit cyclization eliminated the reaction pathway completely. To build upon the ealier studies of metal cationized peptides, and to determine the extent to which changes of the size of the cyclic intermediate may influence the tendency to form sequence ions, our group recently investigated and reported on the incorporation of “alternative” amino acids such as β-alanine (βA), γ-aminobutyric acid (γABu), εcaproic acid (Cap), and 4-aminomethylbenzoic acid (4AMBz) into the sequence of model protonated peptides, and the effect(s) of these residues on relative product ion intensities [39] (figure 1.3). For protonated peptides, the position of the “alternative” amino acids in XAAG, AXAG, and AAXG (where X represents the position of the “alternative” amino acid) had a significant influence on the CID spectrum by inhibiting or completely suppressing the formation of specific bn+ and yn+ ions. This effect was attributed to the 7 prohibitive effect of forcing cyclization and intramolecular nucleophilic attack to progress through larger cyclic intermediates, which would be kinetically slower to form and entropically less favored, when amino acids such as βA, γAbu or Cap were used. Cyclization was prohibited when the 4AMBz residue was used because the rigid aromatic ring separates the nucleophile from the electrophilic site of attack. While investigating the CID of protonated XAAG, AXAG and AAXG peptides we found that while the 4AMBz residue tended to eliminate specific bn+ and yn+ ions because the aromatic ring precluded cyclization and intramolecular nucleophililc attack, it also enhanced the formation of other bn+ ions. For example, increased intensities of b3+ and b2+ ions were observed for the peptides A(4AMBz)AG and (4AMBz)AAG, respectively, suggesting that 4AMBz enhances formation of b type product ions that arise via cleavage of the amide bond one sequence position to the C-terminal side of the residue. The positive effect was explained by formation of a highly conjugated oxazolinone species, with the aromatic ring as a substituent. Therefore, the objective of the second study was to extend our earlier experiments and determine the influence of 4AMBz on the formation of the rival (b3-1+cat)+ and (b3+17+cat)+ products from protonated and metal cationized tetrapeptides. For this study peptides with general sequence A(4AMBz)AX and A(4AMBz)GX, where X = G, A, and V, were synthesized. The CID of protonated and metal cationized (Li+, Na+ and Ag+) forms of these model peptides was then examined by multiple-stage ion trap tandem mass spectrometry. The specific hypothesis tested was that the presence and position of the 4AMBz residue would enhance formation of the (b3-1+cat)+ ion, primarily through 8 generation of the stable, highly conjugated oxazolinone, at the expense of the normally favored (b3+17+cat)+ species. The objective of the third group of experiments was to determine the effect on fragmentation patterns of changing the size of the putative cyclic intermediate formed during the nucleophilic attack. In this third study, the influence of βA, γAbu and Cap on the tendency to form (b3-1+cat)+ products from Li+, Na+ and Ag+ cationized AAXG was investigated. The initial hypothesis was that the potential prohibitively large cyclic intermediates would suppress formation of (b3-1+cat)+, as was observed for the protonated versions of the peptide. However, the metal cations may coordinate with the peptide through interactions with multiple amide carbonyl O atoms, and thus kinetically assist formation of a reactive configuration from which nulceophilic attack occurs. As we show here, (b3-1+cat)+ is a prominent, if not dominant, reaction product from the metal cationized AAXG. 9 Scheme 1a O H N H+ H2N O H N N H O Scheme 1b OH O H2N N H O H2N OH O O H O N H O + N O H2N OH O H N N H O H O + O cat+ O N N+ H H O O H2N cat+ H H N+ H O OH O O H N H2N O O H N O H+ N H cat+ O H N N H O Proton transfer H N H N H2N O O O OH H N H2N H2N O cat+ OH O N H O O N H N+ CO, NH=CH2 H O O H N b3+ H N H2N O cat+ O N H OH O (b3+17+cat)+ Scheme 1 Pathway to (b3)+ (1a) and (b3+17+cat)+ (1b) from AAAG through 5membered cyclic intermediates. 10 100 R. I. (%) AA(βA)G (y2) 80 60 40 (b3) 20 0 100 100 125 150 200 225 275 300 AA(γAbu)G 60 40 (y3) 20 0 100 100 125 150 80 R. I. (%) 250 (y2) 80 R. I. (%) 175 (y3) (y2) 175 200 225 250 275 300 AA(Cap)G 60 (y3) 40 20 0 125 100 R. I. (%) 80 60 150 175 200 225 250 275 300 (y3) (y2) 325 350 AA(4AMBz)G (b2) 40 (b3) 20 0 120 140 160 180 200 220 240 260 280 300 m/z (used with permission from reference 39). Figure 1.3 CID spectra of protonated AAXG. 11 320 340 360 CHAPTER II Experimental Methods Mass Spectrometry Wilhelm Wien laid the foundation for the development of mass spectrometry in 1898, when he discovered that charged particles could be deflected by a magnetic field. In experiments conducted between 1907 and 1913, J.J. Thomson studied the deflection of a beam of positively charged ions using combined electrostatic and magnetic fields. The two fields were oriented in such a way that the ions were deflected through small angles in two perpendicular directions: ion trajectories under the influence of the respective fields produced a series of parabolic curves on photographic plates. Each parabola corresponded to ions of a particular mass-to-charge ratio and specific position of each ion depended on its velocity [40]. Modern mass spectrometry is an analytical technique used to measure the massto-charge ratio (m/z) of ions. Mass spectrometry is useful for quantification of atoms and molecules. Because molecules usually have distinctive fragmentation patterns that provide structural information, mass spectrometry is also used for determining chemical and structural information about molecules [41]. In an extension of the work by Wien and Thomson, m/z values are determined by monitoring signal at a detector as a function of some applied electric or magnetic field. The applied field causes a separation that is dependent on m/z value. The ion separation power of a mass spectrometer is described by its resolving power, which is defined by the equation: R = m/∆m 12 where m is the ion mass and ∆m is the difference in mass between two resolvable peaks in a mass spectrum. A typical mass spectrometer comprises of three parts: an ion source, a mass analyzer and a detector as shown in figure 2.1. In this study the focus was on use of Electrospray ionization (ESI) and Quadrupole Ion Trap (QIT) as ionization and mass analysis methods, respectively. Ion source Analyzer Ion Detector Inlet system Sample introduction. Data system Figure 2.1 The components of a mass spectrometer. 13 Electrospray Ionisation Electrospray ionization ( ESI ) is a method used in mass spectrometry that is especially useful in producing ions from macromolecules because it overcomes the tendency of these molecules to fragment when ionized. The phenomenon of electrospray has been known for over hundred years. Chapman in the 1930’s carried out experiments using electrospray ionization. Some 30 years later (1960), the pioneering work of Malcolm Dole et al demonstrated the use of electrospray to ionize intact chemical species. A further 20 years elapsed until work in the laboratory of John Fenn demonstrated for the first time the use of ESI for the ionization of high mass biologically important compounds and their subsequent study by mass spectrometry. This work was to win John Fenn a share of the 2002 Nobel Prize for chemistry. Fenn and co-workers in 1980’s successfully demonstrated the basic principles and methodologies of the ESI technique, including soft ionization of involatile and thermally labile compounds, multiple charging of proteins and intact ionization of complexes. ESI-MS is now a basic tool used in probably every biological chemistry in the world [42, 43]. In electrospray ionization, the analyte is introduced to the source in solution from syringe pump. Flow rates are characteristically in the order of 1µl/min to 5µl/min. The liquid is pushed through a very small charged metal, capillary. The potential difference with respect to the counter electrode is in the range of 2.5 to 5kV. This liquid contains the substance which is to be studied, the analyte, dissolved in a solvent, which is generally more volatile than the analyte. Volatile acids or buffers are always added to this solution. The analyte exists as an ion in solution either in protonated form or as an anion. As like charges repel, the liquid pushes itself out of the capillary and forms a mist of aerosol of 14 small droplets about 10µm in diameter. This jet of aerosol droplets is sometimes produced by a process involving the formation of a Taylor cone and a jet from the tip of this cone. A neutral carrier gas, such as N2 is sometimes used to desolvate the liquid that is to assist in evaporation of the neutral solvent in the small droplets. As the evaporation occurs, the droplets shrink until they reach the point that the surface tension can no longer sustain the charge, this is referred to as the Rayleigh limit. This is the limiting droplet size at which self-fragmentation occurs with static charge droplets generated in ESI or other ionization processes. At this limit the proximity of the molecules becomes unstable as the similarly charged molecules come close together and the droplets once again explode. This explosion is referred to as coulombic fission because it is the repulsive coulombic forces between charged analyte that influence it. This process repeats itself until the analyte is free of solvent and is a lone ion. The lone ion then continues along to the mass analyzer of a mass spectrometer. In most mass spectrometers, the ESI process is carried at atmospheric pressure. In electrospray process the ions observed are ionized by the addition of a proton (hydrogen ion) to give [M+H]+ (M= analyte molecule, H= hydrogen ion). In other cases a metal cation such as sodium ion is used [M+Na]+ or the molecule can be deprotonated [M–H]-. Also in electrospray, multiply charged ion such as [M+2H]2+ are often observed. Electrospray ionization is a very soft ionization as very little residual energy is retained by the analyte upon ionization. This is why ESI-MS is such a significant technique in studying non-covalent gas-phase interactions. The electrospray process is capable of transferring liquid phase non-covalent complexes into the gas-phase without disrupting their non-covalent interactions. 15 Spray needle tip Rayleigh limit at this point ++++++ ++++++ +++++ Solvent evaporation +++ Coulombic explosion. Multiply charged droplet +ve Power supply -ve + + + Analyte ions Counter electrodes Figure 2.2 Principle of electrospray ionization. 16 Quadrupole Ion-trap Analyzer A quadrupole ion-trap is a sensitive and versatile component of the mass spectrometer. The quadrupole ion-trap (QIT) was developed by the third Nobel Prize winning mass spectrometry pioneer, Wolfgang Paul. His work in the early 1950’s led to the development of the basic parameters of today’s benchtop instruments [44]. However it took breakthroughs in design at the Finnigan MAT in the 1980’s to make the quadrupole mass spectrometer, the simple to use instrument it is today. The quadrupole ion-trap mass analyzer used in this study consists of three hyperbolic electrodes: the ring electrode and the entrance and exit end-cap electrodes. Various voltages are applied to these electrodes which results in the formation of a cavity in which the ions are trapped. Ions produced by electrospray ionization are focused using an octupole transmission system into the ion-trap. Ions may be channeled into the trap through the use of pulsing lens or through a combination of rf potentials applied to the ring electrode. The pulsed transmission of ions into the trap distinguishes ion-traps from “beam” instruments such as quadrupole where ions continually enter the mass analyzer. The time during which ions are allowed into the trap termed the “ionization period”, is set to maximize signal while minimizing space-charge effects. The space-charge effects arises due to too many ions in the trap which distort the electric fields, leading to considerably impaired performance. The ion-trap is kept at a pressure due to the presence of helium. The usual pressure inside the trap is between 10-3 – 10-6 torr. Collision with helium gas present in the ion trap reduce the kinetic energy of the ions and serve to quickly focus trajectories toward the center of the ion-trap, enabling the trapping of the injected ions. 17 Trapped ions are further focused toward the center of the trap through the use of an oscillating voltage, called the fundamental rf , applied to the ring electrode. An ion will be stably trapped depending upon the values for the mass (m) and charge of the ion (e), the radial size of the ion trap (r), the oscillating frequency of the fundamental rf (ω), and the amplitude of the voltage on the ring electrode (v). The reliance of ion motion on these parameters is described by the dimensionless parameter qz qz= 4eV/mr2ω2 It is important to note that quadrupole ion trap is concerned with the criteria that govern the stability (or instability) of the trajectory of an ion within the field, that is the experimental conditions that determine whether an ion is stored within the device or is ejected from the device[45, 46]. Ions can be stored in the trap provided that their trajectories fall within a region of stability within the quadupolar field as defined by the Mathieu parameters az and qz. Depending upon the amplitude of the voltage placed on the ring electrode, an ion of a given m/z will have az and qz values that are within the boundaries of the stability diagram and the ion will be trapped. If az and qz values at that voltage fall outside the boundaries of the stability region, the ion will hit the electrode and will be lost. 18 Fundamental rf. ~ Ring electrode Endcap electrodes v Auxiliary ac. Figure 2.3 Basic components of quadrupole ion trap analyzer. 19 Tandem Mass Spectrometry Tandem mass spectrometry MS/MS involves various steps of mass selection or analysis generally accompanied by some form of fragmentation. Tandem mass spectrometry can be effected in space by using different mass analyzer. For example one mass analyzer can isolate the peptide for investigation. A second mass analyzer then stabilizes the peptide ions while they collide with a neutral gas such as helium, causing them to fragment. A third mass analyzer then catalogs the fragments produced from the peptides. Tandem MS can also be done in a single mass analyzer over time as in quadrupole ion trap. This involves the use of one mass analyzer multiple times to perform the task of isolation and fragmentation of the precursor and product ions. There are various methods for fragmenting molecules for tandem MS, including collision induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), Infrared multi photon dissociation (IRMPD) and Black body infrared radiation (BIRD). In this study collision induced dissociation was used to investigate the metal cationized peptides. All CID processes occurring regularly can be separated into one of two categories based mostly on the translational energy of the precursor ion. For organic ions of moderate mass (several hundred Daltons), low energy collisions, common in quadrupole and ion trap instruments occur in the 1-100eV range of collision energy and high energy collision seen in sector and TOF/TOF instruments are in kiloelectrovolt range. In a quadrupole ion trap, the precursor ions are isolated and accelerated by “onresonance” excitation causing collision to occur and product ions are detected by 20 consequent ejection from the trap. In on-resonance excitation, the isolated precursor ion is excited by applying a small a.c potential across the endcap electrodes, corresponding to the secular frequency of the ion. Ion activation times the order of tens of milliseconds can be used without considerable ion loses, therefore collisions can occur during the excitation period. Because of the time scale, this excitation technique falls in the category of so called “slow heating” processes. However, excitation in an ion trap is still fairly fast, due to the high pressure of the helium present in the trap (≈ 10-3 Torr). For low collision energies, excitation is mostly vibrational since the interaction time coincides with a bond’s vibration period [47]. Multiple stages of CID can be performed in ion trap instrument, although the experiment is restricted to the product ion scan. In product ion scan, the mass analyzer is used to select the user-specified sample ions arising from a particular component, usually the molecular ion ([M+H]+, [M-H]- or [M + cat]+). These chosen ions collide with neutral gas molecules in this case helium which cause fragment ions to be formed (figure 2.4). These product ions are isolated and fragmentation can be performed resulting in MS3 spectrum (figure 2.5). This process can be repeated a number of times, resulting in a series of MSn spectra where ‘n’ represents the number of times the isolationfragmentation cycle has been carried out. All the fragment ions arise directly from the precursor ions specified in the experiment, and this produces a fingerprint pattern specific to the compound under investigation. This is useful for the elucidation of fragmentation pathways and for the identification of molecular structures of the ions. 21 Ion source Mass analyzer MS I Detector Ion source Mass analyzer m/z MS . .. .......... ....... I m/z Detector Collision cell MS/MS I m/z Figure 2.4 Collision induced dissociation of precursor ions. 22 Intensity [M+X]+ Product ions Isolate and fragment intensity Isolate and fragment m/z Figure 2.5 Principle of Tandem mass spectrometry 23 Sample Preparation Procedures Peptides VAAF, FGGFL, AGGFL, LGGFL,FGGGL,YGGFL,VGVPAG, FGGAL, GGGA, AAAG, (4AMBz)AAG, A(4AMBz)AX and A(4AMBz)GX used in this study were generated by solid-phase synthesis methods using Wang resin, conventional coupling procedures [53] and Fmoc-protected amino acids in a custombuilt, multiple reaction vessel peptide synthesis apparatus(figure 2.6). Metal nitrates LiNO3, NaNO3, AgNO3, TlNO3 and Glycine (G), alanine (A), and valine (V)-loaded Wang resins, Fmoc-alanine (Ala), β-alanine [NH2CH2CH2CO2H, βA], γ-aminobutyric acid [NH2CH2CH2CH2CO2H, γAbu], γ-aminobutyric acid labeled with deuterium at the α-carbon position [NH2CH2CH2CD2CO2H, α-d2-γAbu], ε-aminocaproic acid [NH2CH2CH2CH2CH2CH2CO2H, Cap] and 4-aminomethylbenzoic acid [NH2CH2C6H4CO2H, 4AMBz] were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. Peptides, once cleaved from the resin using trifluoroacetic acid, were used without subsequent purification in the CID studies. Solutions of each peptide were prepared by dissolving the appropriate amount of solid material in 1:1(v/v) mixture of HPLC grade MeOH and de-ionized H2O. Equimolar metal nitrate solutions were prepared in deionized H2O to produce the required final concentration. To investigate the dissociation of AAXG peptides for which exchangeable H atoms were replaced with D, the peptide was incubated in a mixture (50:50 by volume) of D2O and CH3OD (Aldrich Chemical, St. Louis MO) overnight prior to analysis. Solutions for ESI experiments were prepared by combining the peptide and metal nitrate solutions in 1:1 ratio. ESI mass spectra were collected using a Finnigan LCQDecaTM ion–trap mass spectrometer (Thermoquest Corporation, San Jose, CA, USA) 24 Mixtures (1:1 v/v) of metal nitrate and peptide, prepared by combining respective stock solutions were infused into the ESI–MS using the incorporated syringe pump at flow rate of 5µl/min. The atmospheric pressure ionization stack settings for the LCQ (lens voltages , quadrupole and octapole voltage offsets) were optimized for maximum (M + cat)+ ion transmission to the ion-trap mass analyzer using the auto-tune routine within the LCQ tune program. Following the instrument tune, the spray needle voltage was maintained at +5kV, the N2 sheath gas flow rate at 25 units was used. This is set arbitrary for the LCQ Deca system. The capillary temperature for this experiment was set at 2200c. This served as desolvation temperature to convert ions from solution phase to gas phase. The ion-trap analyzer was operated at ≈ 1x 10-6 Torr. Helium gas, admitted directly into the ion-trap, was used as the bath/buffer gas to improve the trapping efficiency and as the collision gas for CID experiments. The metal cationized peptides were isolated using a width of 1.5 m/z units. To induce collision activation, the activation amplitude for CID was set between 10 to 30% of 5V. The activation amplitude defines the amplitude of the RF energy applied to the end caps electrodes to effect dissociation. The Q setting used to adjust the frequency of the RF excitation voltage was set at 0.3. The activation time employed at each CID stage was set at 30ms. Spectra were generated by plotting relative intensity to the most abundant ion as a function of m/z. Energy resolved CID profiles generated by scanning the activation amplitude were corrected using the following equations: (AA/30) x ( 0.4 + ( 0.002 x Precursor ion mass ) CAA= (AA/30) x ( 0.4 + ( 0.002 x Precursor ion mass )/(3N-6). 25 Where CAA is the corrected activation amplitude, AA is the normalized collision energy and N is the degrees of freedom. The corrected activation amplitude scale was arbitrarily chosen to facilitate the comparison and measurement of the relative product ion abundances with minimal bias that might originate due to the differences in precursor ion mass of peptides examined here. This allowed a comparison of the relative collision energies/voltages necessary to induce formation of (b3-1+cat)+ and (b3+17+cat)+ for peptides with and without the 4AMBz residue. For ion isolation experiments to probe D for H back exchange, precursor ions were isolated using an isolation width of 0.9 – 1.1 m/z units, which was sufficient to isolate a single isotopic peak. The normalized collision energy was set at 0% (i.e. no imposed collisional activation), the activation Q at 0.30, and the isolation time altered from 10 msec to 1 second. During the isolation period the ions may collide with H2O, which is present as a contaminant in the ion trap and because of its use as solvent for ESI. The check for D for H back exchange was done because such a process could effect the splitting of (b3-1+cat)+ products into distinct isotopic peaks (vide infra), which was the principal feature used to identify and measure the extent of transfer of α-carbon position H or D atoms during fragmentation reactions. 26 Figure 2.6 Multiple reaction vessel peptide synthesis apparatus. 27 CHAPTER III Collision Induced Dissociation of Tl+-cationized peptides Introduction As noted in the introductory chapter, tandem mass spectrometry of cationized peptides and proteins in the gas phase is commonly used to determine the sequence of peptides and identity of the proteins. This information is dependent on the number and identities of the cationizing species and their interactions with the peptide or protein. The fragmentation behavior of protonated peptides has been extensively examined. However, complementary fragmentation and or specific sequence information may be obtained by performing collision induced dissociation experiments on metal cationized peptides. For example, collision induced dissociation (CID) of sodiated peptide leads to fragmentation adjacent to the C-terminal residue, while protonated peptide usually fragments at various places along the peptide backbone [27]. Recent studies have demonstrated significant differences in the CID spectra of protonated and metal cationized peptides [27-38]. An important distinction between the CID spectra generated from protonated peptides and those derived from metal cationized peptides is the preferred formation in the latter of an N-terminus containing (b-type) rearrangement ion labeled (bn+17+cat)+ or (bn+OH+cat)+. While for the protonated peptides, sequence specific b- and y- ions are often the dominant fragment ions in the low energy CID spectra. Studies in our laboratory have shown distinct differences between Ag+ and alkali metal (Li+, Na+) cationized peptides [27], including the fact that the most prominent species observed during the CID of alkali metal(Li+, Na+) cationized leucine enkephalin 28 are the (bn+17+cat)+ ions, while Ag+ cationized peptides favor formation of (bn-1+Ag)+ over both the (bn+17+Ag)+ and (an-1+Ag)+ ions. Similar to the alkali cationized peptides, multi-stage CID of (b2-1+Ag)+ produces prominent (a2-1+Ag)+ ions. While the differences between Ag+ and alkali-metal (Li+, Na+) ions may be due, in part to differences in polarizing power and hardness of the cations, testing of this hypothesis is difficult because of the lack of monovalent transition metal ions to compare to Ag(I). In this set of experiments the multi-stage CID of Tl+ cationized peptides was investigated and compared to those of Ag+, Na+, and protonated analogues. The main goal was to determine whether Tl+ cation would demonstrate reaction pathway similar to alkali metal cations or Ag+. Result and Discussion CID of protonated and metal cationized VAAF, YGGFL and VGVAPG For the sake of illustration and comparison, figure 3.1 shows the CID spectra of protonated, sodiated, argentinated and thallium cationized VAAF. The major peaks observed in spectra of protonated VAAF were b3+ (242) and y2+ (237) product ions. The difference in m/z values between the (M+H)+ and b3+ is 165u (407-242) thus identifying phenylalanine (147u) and H2O (18u). The ∆m/z value resulting in the formation of y2+ (237) was 170u which is consistent with the loss of N-terminus residues valine (99u) and alanine (71u). For Na+ cationized VAAF, the peak with the largest m/z value was (b3+17+Na)+ (282u). The amino acid cleaved from the C-terminus was identified as phenylalanine (147u), since (M+Na)+ had an m/z value of 429u. The Ag+ cationized 29 version generated (b3-1+Ag)+ (348,350) as the major product ion more than (a3-1 + Ag)+ (320,322) and (b3 + 17 + Ag)+ (366,368) species. Silver ion has two stable isotopes 107Ag and 109Ag of almost equal abundance and the product ion spectra shown are those of the two isotopes. In this survey, the most prominent product ion resulting from the CID of Tl+ cationized VAAF was (b3+17+Tl)+ having a mass unit of 462u and 464u due to two isotopes of thallium (205Tl and 207Tl). Other product ions observed were, (b3-1+Tl)+ (444, 446) and (b2-1+Tl)+ (373, 375). One striking observation is that, among the metal cationized peptide (VAAF) investigated Tl+ cationized peptide generated greater abundance of (b2-1+Cat)+ ions. The most prominent product ion generated from the CID of protonated YGGFL figure 3.2, was b4+ (425u) ion due to the neutral loss of leucine (113). The other product ion observed was a4+ (397u). In contrast to CID of protonated peptide, CID of Na+ cationized YGGFL generated (b4+17+Na)+ in abundance. Other minor products obtained were (b4-1+Na)+ and (a4-1+Na)+ ions. The remarkable differences between the protonated peptides and Na+ cationized versions can be attributed to the difference in the binding sites within the peptide. The dissociation of alkali cationized peptides is proposed to involve the binding of the alkali metal ion to amide carbonyl atoms, rather than amide nitrogen atoms [38]. In accord with earlier studies, triplet of product ions, consisting of (b4-1+Ag)+, (a4-1+Ag)+ and (b4+17+Ag)+ were observed in the CID spectra of Ag+ cationized YGGFL. It is interesting that the yield of (b4+17+Ag)+ ion from YGGFL was approximately 35%(figure 3.2c), significantly higher than the corresponding ions 30 (b3+17+Ag)+, generated from VAAF (figure 3.1c). This may be due to the beneficial interaction between the metal ion and the aromatic ring at the second amino acid, in combination with the size of the peptide, to make the C-terminal rearrangement reaction favorable. Similar to Na+ cationized versions, the most prominent product ion resulting from the CID of Tl+ cationized leucine enkephalin was (b4+17+Tl)+ ion (figure 3.2d). This is in contrast to the major product ion observed in the CID of (Tl + VGVAPG)+ (figure 3.3d), in which (b4-1+Tl)+ had the highest peak intensity. This trend in which the major product ion was either (bn+17+Tl)+ or (bn-1+Tl)+ was also observed with other Tl+ cationized peptides investigated in this study. Figure 3.3 shows the CID spectra of VGVAPG in (a) protonated, (b) Na+, (c) Ag+ and (d) Tl+ cationized forms. For this peptide, each cation demonstrated a unique pattern of fragmentation. One interesting observation was that there was high abundance of (a4-1+Tl)+ compared to (a4-1+Ag)+ among the two metal cationized peptides. The spectra shown in figures 3.1-3.3 are representative, of the spectra generated from majority of the peptides included in this study. The study revealed that unlike the Ag+ cationized versions, CID of the alkali cationized (Li+, Na+) versions of the peptides did not generate prominent (bn-1+cat)+ ions. The different behaviors of Ag+ and Na+ metal ions are strikingly consistent with their differences in hardness as lewis acids, Na+ prefers to interact with the harder O (oxygen) donors, whereas Ag+ prefers to bind to softer N (nitrogen) donors. It is interesting then, that generation of (bn-1+cat)+ which involves transfer of proton ultimately to an N- atom is the preferred pathway for Ag+ cationized peptides. The loss of H2O, which involves 31 transfer of a proton instead to an O- atom is the preferred pathway for the alkali cationized (Li+, Na+) peptides. The CID of Tl+ cationized peptides (MS/MS) resulted in the formation of either (bn+17+Tl)+ or (bn-1+Tl)+ (figures 3.1-3.3) product ions . It was found that the tendency to produce (bn-1+Tl)+ versus (bn+17+Tl)+ products depended significantly on peptide sequence (Tables 1 and 2). With several peptides, the product ion distribution generated from Tl+ cationized species were most similar to those generated from group I metals, while for others the distribution was more similar to those observed for protonated and Ag+ cationized peptides. These observations suggest that a straight forward relationship between cation hardness and reaction pathway, will be difficult to identify. Multiple-stage CID of Tl+ cationized product ions MSn experiments were also conducted to elucidate the fragmentation pathways for the dissociation of (bn+17+Tl)+ or (bn-1+Tl)+ product ions, (figures 3.4-3.7). The MS3 product ion spectra (3.4(b)-3.7(b)) were generated by isolating and dissociating the most abundant peak in the MS/MS spectra (3.4(a)-3.7(a)). Subsequent MSn spectra were then generated by isolating and dissociating the most abundant peaks resulting from the prior CID stage (MSn-1). The collision activation of (b4+17+Tl)+ (597) from (LGGFL+Tl)+ generated a new product ion (b3+17+Tl)+ (450) via the loss of 147u which is consistent with the neutral loss of F (phenylalanine) from the C-terminus. Subsequent CID of the (b3+17+Tl)+ (450) product ion (MS4) resulted in the loss of 75u and 245u consistent with the loss of (glycine+ H2O) and elimination of the metal ion to form (b2-1+Tl)+ (374.9) and Tl+ (205) ions as the major product ions which is in contrast to the behavior of Ag+ or 32 alkali cationized versions. This reaction pattern was also observed with the multi stage CID of (FGGLL+Tl)+ peptides (figure 3.5). As illustrated in figure 3.6, the dissociation pattern of (FGGGL+Tl)+ was unique in that the CID of (b4+17+Tl)+ having mass unit of 541 resulted in the production of (b3-1+Tl)+ (466) and Tl+ (205) as the major product ions. In most of the peptides investigated, the multi stage CID of the thallium product ions resulted in the high yield of the bare metal cation (Tl+) signifying a weak binding between the metal and the peptide. Inspection of the data showed a striking influence of the sequence of the peptide on the relative intensities of the (bn+17+cat)+ and (bn-1+cat)+ product ions. For instance, at the same corrected activation amplitude,(spectra not shown), CID of Ag+ cationized GGG and GGA produced (bn+17+Ag)+ product intensities of 8% and 45% respectively, indicating an increase in intensity as the side group of the C-terminal amino acid become bulky. This is in accord with earlier studies by Glish and coworkers. However, in contrast to their studies, the influence of the N-terminal amino acid was also observed. For example, FGG and YGG produced higher relative intensities of (bn+17+cat)+ and (bn-1+cat)+ product ions than GGG. It is apparent that the N-terminal amino acid lies on neither the five-membered ring during the C-terminal rearrangement nor that formed during the N-terminal rearrangement, and thus is not expected to play a significant role in the stability of the rings or influence the probability of their formation. In this case the bulky group at the N-terminus influences the binding of the metal ion, and in particular increases the probability that the metal ion can migrate to the cleavage site to polarize the carbonyl bond. Indeed, the yield of the (b2+17+cat)+ and (b2 -1+cat)+ product 33 ions are significantly higher for FGG and YGG, with the aromatic side groups capable of coordinating the metal ion and altering the metal ion binding position on the peptide. Summary In this study, the CID results showed that the tendency to produce (bn+17+Tl)+ or (bn-1+Tl)+ depended significantly on the peptide sequence. The survey demonstrated that MSn of the protonated versions of the peptides failed to give the accurate sequence information. The sequencing failed because of the tendency to eliminate amino acids from either the N- or C-terminus, leading to an ambiguity in sequence determination. It was observed that multistage CID (MSn) of Ag+ and alkali metal cationized peptides can lead to unambiguous determination of the sequence of peptides (Scheme 6 and 8), whereas Tl+ cationized versions are not preferable. This is because at higher CID stages of the latter, the reaction channel leading to the elimination of the metal cation is more preferred (scheme 2-5) than the formation of (bn) or (an) sequence ions which is observed with former species. One interesting observation was that among the metal cations investigated, Tl+ cationized peptides tended to produce the highest yield of (yn-1+cat)+ species. The appearance of the prominent (yn-1+cat)+ ion may be attributable to the metal ion affinity for this product ion and is being explored further in our research work. 34 100 R. I. (%) 75 (a) 237 y2+ (H+) VAAF 242 b3+ 50 b2+ 171 y1+ 166 25 0 125 150 y3+ 308 197 175 200 225 250 275 300 -H 2O 389 361 325 350 375 400 425 100 25 0 125 150 175 200 264 236 193 211 225 282 (b3+17+Na)+ (b3-1+Na)+ 50 (a3-1+Na)+ (b2-1+Na)+ R. I. (%) (b2+17+Na)+ (b) (Na+) 75 250 275 300 325 350 375 400 425 450 100 R. I. (%) 75 (c) (Ag+) (b2-1+Ag)+ 50 (a3-1+Ag)+ 320,322 (a2-1+Ag)+ 249,251 0 100 125 150 175 200 225 250 275 300 100 R. I. (%) (b3+17+Ag)+ 366,368 277,279 25 75 (b3-1+Ag)+ 348,350 (d) (Tl+ ) (b2-1+Tl)+ 373,375 325 350 (b3-1+Tl)+ 444,446 375 400 425 450 475 500 525 600 625 462,464 (b3+17+Tl)+ 50 25 0 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 m /z Figure 3.1 CID spectra of protonated and metal cationized VAAF 35 100 (a) R. I. (%) 75 YGGFL 425 b4+ (H+) a4+ 397 50 538 y3+ 336 25 510 0 225 275 300 325 350 375 400 425 450 475 50 (b3+17+Na)+ 318 25 (b4-1+Na)+ (Na+) 500 525 550 575 600 550 575 600 625 650 675 775 800 465 (b4+17+Na)+ (b) 75 R. I. (%) 250 (a4-1+Na)+ 100 419 447 0 225 100 R. I. (%) 75 250 275 300 325 350 375 400 (c) 425 450 475 500 525 531,533 (b4-1+Ag)+ (Ag+) 50 503,505 (a4-1+Ag)+ (b3-1+Ag)+ 384,386 25 549,551 (b4+17+Ag)+ 0 350 375 400 425 450 475 (d) (Tl+) 50 (b3-1+Tl)+ 480,482 25 500 (a4-1+Tl)+ R. I. (%) 75 325 (b3+17+Tl)+ 498,500 525 (b4-1+Tl)+ 300 100 550 575 600 645,647 (b4+17+Tl)+ 627, 599, 629 601 0 400 425 450 475 500 525 550 575 600 625 650 675 700 725 m /z Figure 3.2 CID spectra of protonated and metal cationized YGGFL. 36 750 100 327 b4+ (a) R. I. (%) 75 (H+) a4+ 299 50 a3+ 228 211 173 25 VGVAPG b3+ 256 282 b5+ 424 -H2O 481 0 250 (a4-1+Na)+ 100 (b) R. I. (%) 75 (Na+) 50 275 321 25 300 325 350 375 400 425 349 367 450 475 500 525 (b5+17+Na)+ 225 (b5-1+Na)+ 200 (b4+17+Na)+ 175 (b4-1+Na)+ 150 464 -H2O 503 446 0 275 300 325 350 375 (Ag+) 50 25 405,407 225 250 275 300 325 350 375 400 500 525 550 (M+Ag)+ 433,435 605,607 (a3-1+Tl)+ 50 Tl+ 203,205 450 475 500 501,503 529,531 (b3-1+Tl)+ (Tl+) 425 (a4-1+Tl)+ (d) R. I. ( %) 475 458, 430, 460 432 525 550 (b5+17+Tl)+ 200 100 25 450 362,364 0 75 425 (b4-1+Tl)+ R. I. (%) (b3-1+Ag)+ (c) 75 400 (b4-1+Ag)+ 250 (a4-1+Ag)+ 225 100 575 600 625 -H2O 683,685 644, 646 0 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 m /z Figure 3.3 CID spectra of protonated and metal cationized VGVAPG. 37 YGGFL Product ions (% R.I) M-H2O b4+17 b4 b3+17 b3 Tl 8 100 34 2 4 H 22 0 100 0 2 Ag 1 27 100 0 2 Na 0 100 6 0 0 Product ions (% R.I) M-H2O b5+17 b5 b4+17 b4 Tl 56 19 12 11 100 H 2 0 3 0 100 Ag 5 0 6 0 100 Na 12 100 14 18 30 WHWLQL Product ions (% R.I) M-H2O b5+17 b5 b4 y4 Tl 21 100 61 17 14 H 42 0 100 73 36 Ag 7 8 100 26 2 Na 8 100 16 2 3 Table 1 representative product ion distributions. VGVAPG 38 GPA Product ions (% R.I) M-H2O b2+17 b2 a2 y1 Tl 100 15 47 0 39 H 18 0 100 8 0 Ag 55 83 30 8 74 Na 100 57 21 0 5 FGG Product ions (% R.I) M-H2O b2+17 b2 a2 a1 Tl 21 7 34 0 79 H 1 0 100 0 0 Ag 9 21 100 37 0 Na 60 52 100 0 0 GHG Product ions (% R.I) M-H2O b2+17 b2 b1 -35u Tl 100 0 34 0 0 H 83 0 81 26 100 Ag 2 0 100 0 0 Na 56 0 100 0 0 Table 2 representative product ion distributions of protonated and metal cationized GPA, FGG and GHG peptides. 39 100 597 (b4+17+Tl)+ (a) 80 (b4-1+Tl)+ 60 579 40 (a4 CID of 710 -1+Tl)+ (-H2O) 692 551 20 0 (%) 0 100 Relative intensity 100 200 300 400 500 600 700 800 (b3+17+Tl)+ 450.1 (b) 80 CID of 710_597 60 40 Tl+ 20 205 (b3-1+Tl)+ 432 (b4+17+Tl)+ 596.8 0 0 100 100 300 400 (Tl+) 205 (c) 80 200 500 600 700 800 (b2-1+Tl)+ 374.9 CID of 710_597_450 60 (b3+17+Tl)+ 450 40 20 0 0 100 200 300 400 500 600 700 800 m/z Figure 3.4 Multi-stage CID of (LGGFL+Tl)+ product ions. (M+Tl)+ 710 692 MS/MS 579 -113 (L) (b4-1+Tl)+ 597(b4+17+Tl)+ MS3 -147 (F) 450.1(b3+17+Tl)+ MS4 -75 (G+H2O) 205(Tl)+ Scheme 2 374.9(b2-1+Tl)+ Multi-stage CID scheme of thallium cationized LGGFL. 40 100 (b4-1+Tl)+ (b4+17+Tl)+ 597 80 60 (an-1+Tl)+ 579 551 (a) 40 CID of 710 20 0 Relative intensity (%) 0 100 200 300 400 500 600 700 800 (b3+17+Tl)+ 484 100 80 60 CID of 597 (b) Tl+ 40 205 20 0 0 100 200 Tl+ 205 100 80 60 300 400 500 600 700 800 (b2-1+Tl)+ 324 409 CID of 484 (b3+17+Tl)+ 466 (c) 40 20 0 0 100 200 300 400 500 600 700 m/z Figure 3.6 Multi-stage CID of (FGGLL+Tl)+ product ions. 41 800 (M+Tl)+ 710 551 (a4-1+Tl)+ MS/MS 579 (b4-1+Tl)+ -133(L) 597 (b4+17+Tl)+ MS3 -113 (L) 205 (Tl)+ 484 (b3+17+Tl)+ MS4 409 (b2-1+Tl)+ 205 (Tl)+ Scheme 3 -75 (G+H2O) MS4 Multi-stage CID scheme of thallium cationized FGGLL. (M+Tl)+ 654 MS/MS 523 (b4-1+Tl)+ -113 (L) 541 (b4+17+Tl)+ 522.9 (b4-1+Tl)+ MS3 -75 (G+H2O) 205 (Tl)+ 466 323.9 205 (Tl)+ Scheme 4 Multi-stage CID scheme of thallium cationized FGGGL 42 (b4+17+Tl)+ 100 80 541 (a) CID 654 60 (b4-1+Tl)+ 40 523 20 0 0 Relative intensity (%) 100 100 200 300 Tl+ 205 (b) 400 600 700 800 (b3-1+Tl)+ 80 466 324 60 500 (b4-1+Tl)+ 522.9 CID 654_541 40 20 0 0 100 200 300 400 500 600 700 800 323.9 100 (c) 80 CID 654_541_466 60 40 Tl+ 437.8 20 205 0 0 100 200 300 400 500 600 700 m/z Figure 3.6 Multi-stage CID of (FGGGL+Tl)+ product ions. 43 800 (b4+17+Tl)+ 100 (a) 80 (b4-1+Tl)+ 60 555 537 CID 668 40 20 668 0 0 100 200 300 400 100 80 600 700 800 408 (b) CID 668_555 60 40 Tl+ 20 537 205 0 0 100 100 80 200 Tl+ 205 (c) 300 400 279.9 60 40 500 600 700 800 (b3-1+Tl)+ 333 (b2-1+Tl)+ Relative intensity (%) 500 (b3+17+Tl)+ 390 CID 668_555_408 20 0 0 100 200 300 400 500 600 m/z Figure 3.7 Multi-stage CID of (AGGFL+Tl)+ product ions. 44 700 800 (M+Tl)+ 668 MS/MS 537 (b4-1+Tl)+ -113 (L) 555 ( b +17+Tl)+ 4 MS3 205 (Tl)+ -147 (F) 408 (b3+17+Tl)+ MS4 -18 (H2O) -75 205 (Tl)+ 390 (b3-1+Tl)+ 333 (b2-1+Tl)+ Scheme 5 Multi-stage CID scheme of thallium cationized AGGFL. (M+Na)+ 487 MS/MS -114 (L) 373 (b4+17+Na)+ MS3 226 -147 (F) (b3+17+Na)+ 168.9 (b2+17+Na)+ MS4 -18 (H2O) 150.8 + (b2-1+Na) 208 (b3-1+Na)+ Scheme 6 Multi-stage CID scheme of sodium cationized AGGFL 45 (M+H)+ 464 MS2 -18 (H2O) 446.1 -131 332.9 (b4)+ -28 MS3 FL 305 (a4)+ MS4 -74 231 -28 MS5 203 (AGG+H)+ MS6 -71 (A) 132 (GG+H)+ Scheme 7 Multi-stage CID scheme of protonated AGGFL 46 (M+Ag)+ 572 -131 MS/MS -113 (L) 459 (b4+17+Ag)+ 441 (b4-1+Ag)+ MS3 -28 412.9 (a4-1+Ag)+ F -119 MS4 293.9 (b3+17+Ag)+ MS5 -28 265.9 (a3-1+Ag)+ MS6 G -29 236.9 (b2-1+Ag)+ Scheme 8 Multi-stage CID scheme of silver cationized AGGFL 47 CHAPTER IV Influence of a 4-aminomethylbenzoic acid residue on competitive fragmentation pathways during CID of metal cationized peptides Introduction As noted in earlier sections, a prominent, if not the preferred dissociation pathway for Li+ and Na+ cationized peptides is formation of (bn+17+cat)+ ions [33,34,48]. Except for a relatively few cases in which peptides contain arginine residues to localize the added proton [49,50], the protonated analog (bn+17+H)+ is rarely observed. The currently accepted mechanism for generation of (bn+17+cat)+ ions is cyclization and intramolecular nucleophilic attack with proton transfer. As in the case of the bn+/(bn-1+cat)+ pathway, the electrophilic site of attack in the (bn+17+cat)+ pathway is the carbonyl carbon of the amide group being cleaved. In this case, however, the nucleophile is the carbonyl oxygen of the C-terminal carboxylic acid [31,35,51,52] (reaction b in scheme 1). For metal cationized peptides in particular, the (bn-1+cat)+ and (bn+17+cat)+ ions are the products of competing reaction pathways that involve cleavage of the same amide bond. Incorporation of “alternative” amino acids such as β-alanine (βA), γ-aminobutyric acid (γABu), ε-aminocaproic acid (Cap), and 4-aminomethylbenzoic acid (4AMBz) into the sequence of a model peptide is known to have a substantial effect on relative product ion intensities [39]. For protonated peptides, the position of the “alternative” amino acids in XAAG, AXAG, and AAXG (where X represents the position of the “alternative” amino acid) [39] inhibits or completely suppresses formation of specific bn+ and yn+ ions. This observation was attributed to the prohibitive effect of forcing cyclization and intramolecular nucleophilic attack to progress through larger cyclic intermediates, which 48 would be kinetically slower to form and entropically less favored, when amino acids such as βA, γAbu or Cap were used. Cyclization was prohibited when the 4AMBz residue was used because the rigid aromatic ring separates the nucleophile from the electrophilic site of attack. For the metal (Li+, Na+ and Ag+) cationized peptides [29] the “alternative” amino acids, when placed at the C-terminus of the model peptide acetyl-FGGX, inhibited the formation of the (b3+17+cat)+, because these amino acids either require larger cyclic intermediates to transfer the required O and H atoms, or prohibited cyclization because of the inclusion of a rigid aromatic ring. During the initial CID study of protonated XAAG, AXAG and AAXG it was discovered that while the 4AMBz residue can cause the disappearance of specific bn+ and yn+ ions because the aromatic ring precluded cyclization and intramolecular nucleophililc attack, formation of other bn+ ions appeared to be enhanced. For example, increased intensities of b3+ and b2+ ions were observed for A(4AMBz)AG and (4AMBz)AAG, respectively, suggesting that 4AMBz enhances formation of b type product ions that arise via cleavage of the amide bond one sequence position to the C-terminal side of the residue. The positive effect was attributed to the fact that the oxazolinone product formed in the dissociation reaction would be highly-conjugated (and thus stable) and feature an aromatic ring substituent. This is shown for protonated A(4AMBz)AG in scheme 9. The overall aim of the experiments described in this chapter was to extend earlier experiments and investigate the influence of 4AMBz on the formation of the rival (b3-1+cat)+ and (b3+17+cat)+ products from metal cationized tetrapeptides. As noted above, these specific ions are generated by competing reactions that ultimately involve cleavage of the same amide bond. For this study, model peptides with general sequence 49 A(4AMBz)AX and A(4AMBz)GX, where X = G, A, and V, were synthesized. The CID of protonated and metal cationized (Li+, Na+ and Ag+) forms of these model peptides was then examined by multiple-stage ion trap tandem mass spectrometry and by probing the change in product ion intensities with applied collision voltage (so called CID or dissociation profiles). The specific hypothesis tested was that the presence and position of the 4AMBz residue would enhance formation of the (b3-1+cat)+ ion, primarily through generation of the stable, highly conjugated oxazolinone, at the expense of the normally favored (b3+17+cat)+ species. Results CID of cationized GGGA and AAAG The structures of the group of peptides synthesized and examined by ion-trap CID are provided in figure 4.1. The first goal of this study was to probe for a general effect of sequence on the formation of bn+, (bn-1+cat)+ and (bn+17+cat)+ ions. To this end the CID patterns for cationized GGGA and AAAG were examined. Figure 4.2 shows the CID (MS/MS stage) spectra derived from GGGA cationized by attachment of H+ (4.2a), Li+ (4.2b), Na+ (4.2c) and Ag+ (4.2d). CID of protonated GGGA produced a prominent peak at m/z 243 via a loss H2O. One possible mechanism by which the m/z 243 product may be generated is the conventional “oxazolone” pathway [17] operative in formation of bn+ and yn+ ions. For generation of the [M-H2O+H]+ ion from GGGA (m/z 243), this pathway would involve cyclization, nucleophilic attack and elimination of H2O from the Cterminal acid group [54]. An alternative route would be a retro-Koch or retro-Ritter 50 type reaction, originally proposed by O’Hair and coworkers, which would involve elimination of an amide carbonyl oxygen atom [28,55,56]. Subsequent CID (MS3 stage, spectrum not shown) of the species at m/z 243 showed the loss of 28 and 29 mass units (u) with the former pathway more prominent than the latter. Loss of 28 as CO is the characteristic decomposition pathway for bn+ ions that feature the oxazolinone ring [19,20]. Loss of 29 has been observed in our laboratory for species in which the H2O molecule is eliminated from an amide position, and in the present case reflects the loss of HN=CH2 from the N-terminal end of the m/z 243 species. The dominant product ion in the CID spectra derived from the alkali metal cationized versions of GGGA was (b3+17+cat)+ at m/z 196 and 212 for Li+ (4.2b) and Na+ (4.2c), respectively. In each case, the rival (b3-1+cat)+ ion appeared at relative intensities of only ~5% (m/z 178 and 194 for Li+ and Na+, respectively). Li+ cationized product ions in particular are prone to the formation of adducts through association reactions with adventitious H2O in the ion trap, and an H2O adduct to the (b3-1+Li)+ species derived from AAAG would be isobaric with (b3+17+Li)+. To test for generation of an H2O adduct, (b3-1+Li)+ was selectively isolated and stored in the ion trap, without imposed collisional activation, for 30 msec after its formation by CID of Li+ cationized AAAG (spectrum not shown): all species other than the one selected for isolation were resonantly ejected from the ion trap. The relative intensity of a peak 18 u higher, indicative of formation of an H2O adduct, was only ca. 5%, relative to (b3-1+Li)+, thus indicating that the species as m/z 196 in figure 4.2b is primarily attributable to formation of (b3+17+Li)+. No H2O adducts to (b3-1+Na)+ were observed when the same test was applied to the Na+ cationized version of AAAG. 51 CID of Ag+ cationized GGGA (4.2d) generated (b3+17+Ag)+ (m/z 296 and 298), (b3-1+Ag)+ (m/z 278 and 280) and (a3-1+Ag)+ (m/z 250 and 252). For the Ag+ cationized version, the (b3+17+Ag)+ ion was observed at a relative intensity of ca. 25%, while the rival (b3-1+Ag)+ product was the base peak. As for the Li+ and Na+ cationized versions of the peptide, isolation of the (b3-1+Ag)+, without imposed collision activation, demonstrated that formation of H2O adducts was not a significant source of error while determining relative (b3+17+Ag)+ peak intensity. Figure 4.3 shows the CID spectra derived from AAAG cationized by attachment of H+ (4.3a), Li+ (4.3b), Na+ (4.3c) and Ag+ (4.3d). For the protonated version of the peptide, b3+ at m/z 214 was the dominant sequence ion generated, with b2+, y2+ and a3* at m/z 143, 147 and 169, respectively, also observed. A shift in preference from b4+ for GGGA to b3+ for AAAG demonstrates that the formation of bn+ ions is, in general, less favored when the cyclization and nucleophilic attack steps occur at or “across” a glycine residue. As shown in figures 4.3b and 4.3c, the change in peptide sequence also influenced significantly the product ion distribution for the Li+ and Na+ cationized versions of AAAG, respectively. While (b3+17+Li)+ at m/z 238 was the most abundant product ion generated from the Li+ cationized version of AAAG, the (b3-1+Li)+ and (a3-1+Li)+ products at m/z 220 and 192 were generated at relative intensities significantly higher than from GGGA. For the Na+ cationized version of AAAG, the (b3-1+Na)+ species at m/z 236 was the most abundant product. For Ag+ cationized AAAG (4.3d), (b3-1+Ag)+ species at m/z 320 and 322 was the dominant product ion, and the rival (b3+17+cat)+ product, which would have m/z values of 338 and 340, was not observed. As with the 52 GGGA peptides, isolation and storage of (b3-1+cat)+ derived from AAAG, without imposed collision activation, showed that H2O adducts did not contribute significantly to the apparent (b3+17+cat)+ peak intensities. CID (MS3 stage, spectra not shown) of (b3+17+Li)+ and (b3+17+Na)+ generated (b2+17+Li)+ and (b2+17+Na)+, respectively, as the dominant product ions. Loss of H2O from (b3+17+Li)+ or (b3+17+Na)+ to generate the (b3-1+cat)+ species was not a prominent fragmentation pathway. This observation further confirms that the (b3+17+cat)+ and (b3-1+cat)+ species represent the products of competing dissociation pathways (from the same precursor ion) that involves cleavage of the same amide bond, rather than two steps along a sequential dissociation pathway. CID of cationized A(4AMBz)AG Comparison of the CID of cationized GGGA and AAAG demonstrate that relative product ion intensities are influenced by the position of the G residue(s) in the peptide sequence, and that formation of product ions that involve cyclization and nucleophilic attack across a glycine residue, such as formation of (b3+17+cat)+ from AAAG or b3+ from protonated GGGA, are less favored compared to a case where the same processes instead involve an alanine residue. The high yield of both (b3+17+cat)+ and (b3-1+cat)+ from Li+ or Na+ cationized versions of AAAG made this system particularly well suited for an examination of the influence of the 4AMBz residue on competition between the two dissociation pathways. Figure 4.4 shows CID spectra derived from A(4AMBz)AG cationized by attachment of H+ (4.4a), Li+ (4.4b), Na+ (4.4d) and Ag+ (4.4e). The sole product ion 53 generated from protonated A(4AMBz)AG was b3+ at m/z 276. Both b2+ and y2+ were generated in relatively high abundance from protonated AAAG, but were not observed in the CID spectrum of protonated A(4AMBz)AG. Elimination of the pathways leading to b2+ and y2+ which would have m/z values of 205 and 147, respectively, is attributed to the fact that the cyclization and nucleophilic attack steps necessary to generate the products are inhibited by the rigid aromatic ring of the 4AMBz residue [39]. Two major product ions were observed following CID of Li+ cationized A(4AMBz)AG: one at m/z 282 and another at m/z 300. The species at m/z 282 is formed via a neutral loss of 75 u and is attributed to (b3-1+Li)+. The species at m/z 300 was initially attributed to (b3+17+Li)+. As noted above, Li+ cationized product ions are prone to the formation of adducts through association reactions, and the H2O adduct to the (b3-1+Li)+ species derived from A(4AMBz)AG would be isobaric with (b3+17+Li)+. The (b3-1+Li)+ species was selectively isolated and stored in the ion trap, without imposed collisional activation, for 30 msec after its formation by CID of Li+ cationized A(4AMBz)AG. As shown in figure 4.4c, isolation and storage of (b3-1+Li)+ lead to formation of an abundant ion at m/z 300, which identifies the latter species as an H2O adduct. The ion at m/z 300 also displayed characteristics of loosely-bound adducts in ion trap mass spectrometers [57-59], including a pronounced tail to the low-mass side of the peak and a chemical mass shift. We therefore conclude that the peak at m/z 300 observed in figure 4.4b is not (b3+17+Li)+, but instead an H2O adduct to (b3-1+Li)+. As shown in figures 4.4d and 4.4e, (b3-1+cat)+ at m/z 298 and 383 was the dominant product ion generated by CID of the Na+ and 109Ag+ cationized versions of A(4AMBz)AG, respectively. Because of lower hydration energy, H2O adducts to Na+ 54 cationized products are rarely, if ever, observed in our ion trap under the experimental conditions used here. To eliminate any ambiguity in the evaluation of CID product ion yields, the (b3-1+Na)+ and (b3-1+Ag)+ product ions were isolated and stored, without imposed collision activation, as described above for the Li+ cationized version. No formation of H2O adducts was observed. The (b3+17+cat)+ product ion at m/z 316 was generated at a relative intensity of ~3% for Na+ cationized A(4AMBz)AG, and was not observed for the Ag+ cationized version of the peptide. Comparison of the spectra shown in figure 4.4 to those in figure 4.3 demonstrates the significant influence of the 4AMBz residue on the product ion intensities, and in particular, the competition between (b3-1+cat)+ and (b3+17+cat)+ from Li+ and Na+ cationized versions of the peptides. CID of Na+ cationized A(4AMBz)AX and A(4AMBz)GX Comparison of the CID spectra in figures 4.2 and 4.3 reveals the sensitivity of relative product ion intensities to the sequence of the model peptides, particularly with respect to the specific positions of G and A residues. To examine further the competition between the (b3-1+cat)+ and (b3+17+cat)+ product ions, the CID of A(4AMBz)AX and A(4AMBz)GX, where X= A or V was investigated. In these experiments, the hypothesis was that the use of larger amino acids at the C-terminus would enhance formation of (b3+17+cat)+, as was observed in the comparison of ion intensities for metal cationized GGGA and AAAG (figures 4.2 and 4.3). For the A(4AMBz)GX series, any enhancement to the yield of (b3+17+cat)+ due to the large C-terminal amino acid could also be amplified by the fact that the G residue adjacent to the C-terminus may make formation of (b3-1+cat)+ less favorable. For these experiments, CID of the [M+Na]+ ions 55 of A(4AMBz)AX and A(4AMBz)GX was examined because the Na+ cationized product ions are not susceptible to the gas-phase H2O addition reactions that plague experiments with Li+ cationized versions. Figure 4.5 shows CID spectra derived from Na+ cationized A(4AMBz)AA (4.5a), A(4AMBz)AV (4.5b), A(4AMBz)GA (4.5c) and A(4AMBz)GV (4.5d). No appreciable increase in the intensity of (b3+17+Na)+ was observed when the C-terminal G residue of A(4AMBz)AG was replaced with A or V. As is apparent in figures 4.5a and 4.5b, the (b3+17+Na)+ ion was generated at relative intensities below 5%, and was nearly identical to the yield of the same species from Na+ cationized A(4AMBz)AG. The relative intensity of (b3+17+Na)+ was markedly higher for the A(4AMBz)GX peptides (figures 4.5c and 4.5d, respectively), and appeared at ca. 10% and 40% for A(4AMBz)GA and A(4AMBz)GV, respectively. We attribute the increase in (b3+17+Na)+ intensity relative to (b3-1+Na)+ observed for the A(4AMBz)GA and A(4AMBz)GV peptides to the fact that the reaction to produce the latter product involves cyclization of a G residue, thus making the pathway less favorable. However, regardless of the sequence and identity of the C-terminal amino acids, for no peptide containing the 4AMBz residue was the intensity of the (b3+17+Na)+ product greater than (b3-1+Na)+. CID of Na+ cationized AA(4AMBz)G and (4AMBz)AAG The spectra shown in figures (4.4, 4.5) demonstrate a significant influence by the 4AMBz residue on the competition between formation of (b3-1+cat)+ and (b3+17+cat)+ from the Li+ and Na+ cationized forms of the model peptides. An important question was 56 whether the influence was due to the specific position of 4AMBz, or instead the mere presence of the residue within the peptide sequence. In a previous CID study of model peptides with “alternative” amino acids at the C-terminal position [39], we observed the complete elimination of the (bn+17+cat)+ pathway for peptides with an aminobenzoic acid residue. To test for an influence by the position of the aromatic residue on dissociation patterns in the present set of experiments, CID of cationzied AA(4AMBz)G and (4AMBz)AAG was examined. For the sake of brevity, only the CID spectra of Na+ cationized AA(4AMBz)G and (4AMBz)AAG are shown in figure 4.6. For Na+ cationized AA(4AMBz)G, the dominant product ion generated was (b3+17+Na)+ at m/z 316. The (b3-1+Na)+ and (a3-1+Na)+ product ions, which would have m/z values of 298 and 270, respectively, were not observed. Similar results were obtained for the Li+ cationized peptide (spectrum not shown). The absence of the (b3-1+cat)+ and (a3-1+cat)+ peaks is consistent with the inhibition, by the 4AMBz residue, of the cyclization step and nucleophilic attack necessary to produce the species along the “oxazolone” pathway. A similar effect was noted in our earlier study of the CID of protonated peptides. For AA(4AMBz)G, the 4AMBz residue clearly influences the competition between the rival pathways by eliminating the (b3-1+cat)+ pathway. For Na+ cationized (4AMBz)AAG, the (b3+17+Na)+, (b3-1+Na)+ and the (a3-1+Na)+ ions at m/z 311, 298 and 270, respectively, were observed with relative abundances similar to those observed in figure 4.3c for Na+ cationized AAAG. For this particular peptide, the 4AMBz residue is located at the N-terminus of the peptide and should not affect formation of (b3+17+Na)+ or (b3-1+Na)+ by impeding the cyclization or nucleophilic attack steps required for generation of these product ions. We can therefore 57 conclude that it is not the mere presence of the 4AMBz that is responsible for an effect on the competition between (b3+17+Na)+ and (b3-1+Na)+, but instead the specific position of the residue in the peptides. Dissociation profiles generated using AAAG and A(4AMBz)AG The observations made using the CID spectra derived from protonated and metal cationzied A(4AMBz)AG clearly demonstrate that the 4AMBz residue influences product ion intensities, and especially the competition between the competing (b3-1+cat)+ and (b3+17+cat)+ products from Li+ and Na+ cationized peptides. The next question that arose was whether the effect of the 4AMBz residue is one of promoting the rate of formation of (b3-1+cat)+, presumably because of the generation of the stable, highly conjugated oxazolinone structure, or instead the suppression or inhibition of the (b3+17+cat)+ pathway. For precursor ions of a given internal energy, the relative peak heights for competing dissociation pathways are dependent on their respective reaction rates [60,61]. To probe in a crude way the response of product ion intensities to changes in internal energy, CID profiles, which show changes in precursor and product ion intensities as a function of the voltage applied to the end-cap electrodes of the ion trap voltage to induce dissociation, were examined. For these specific experiments, the [M+Na]+ ions of AAGV and A(4AMBz)GV were subjected to CID because a prominent (b3+17+Na)+ product was generated from the latter peptide despite the presence of the aromatic amino acid in the sequence. In general, higher collision voltages applied to induce dissociation should lead to higher precursor ion internal energies, but it has been noted that the correlation between 58 collision energies and ion internal energies and their distributions is not necessarily straightforward [61]. Because of uncertainties in ion kinetic energies in ion traps and in the ultimate conversion of collision energy to internal energy, and the need to tune precisely the activation R.F. voltage and frequency, the quadrupole ion trap is less well suited for energy-resolved CID measurements than, for example, guided ion beam instruments. However, the intent in collecting and examining the CID profiles here was not to determine and report the appearance energies or thresholds for the respective dissociation pathways, but instead to probe for gross changes in the relative intensities of (b3-1+cat)+ and (b3+17+cat)+ as a function of the voltage applied to affect CID when the 4AMBz residue is in a sequence position to influence a fragmentation pattern. Figure 4.7 shows the CID profile for Na+ cationized AAGV and A(4AMBz)GV, collected by scanning the normalized collision energy from 10 through 35% (roughly 0.75 → 1.2 V applied). As noted in the experimental section, the normalized collision energy was converted to applied voltage in the laboratory frame of reference, and then divided by the vibrational degrees of freedom (DOF) of the precursor peptide. Using this approach, the rise in product ion intensities can be compared at the similar applied collision voltage per DOF to minimize any erroneous observations and conclusions due to “degrees of freedom” effect on the internal energy needed to induce a fragmentation reaction [61]. For Na+ cationized AAGV, the CID profiles demonstrate that the (b3+17+Na)+ ion is the dominant product at all applied CID voltages beyond which dissociation is observed, and reached a fraction of ion abundance of 0.1 at ca. 0.00960 V/DOF. The rival (b3-1+Na)+ species failed to reach the same fractional abundance of 0.1 even at the 59 highest applied collision voltages. The CID profiles for Na+ cationized AAGV are therefore consistent with a lower critical energy, and thus higher formation rate at a given precursor ion internal energy, for generation of (b3+17+Na)+ compared to (b3-1+Na)+. For Na+ cationzied A(4AMBz)GV, the profiles for both (b3+17+Na)+ and (b3-1+Na)+ are shifted to lower relative collision voltages per DOF primarily because of the greater number of DOF due to the inclusion of the larger 4AMBz residue. The important observation, however, is that for Na+ cationized A(4AMBz)GV, it is the intensity of the (b3-1+Na)+ species that begins to rise at lower relative collision voltages, reaching a fraction of the ion abundance of 0.1 at ca. 0.00617 V. The (b3+17+Na)+ product instead reaches a fractional abundance of 0.1 at 0.00665 V. The decrease of (b3-1+Na)+ ion intensities at higher V/DOF (i.e. above ~.0067) is due to dissociation of (b3-1+Na)+ into (a3-1+Na)+ via the loss of CO. The curve showing the increase in (a3-1+Na)+ intensity as a function of applied CID voltage was not included in figure 4.7 for the sake of clarity. In any case, the CID profile for Na+ cationized A(4AMBz)GV suggests that the critical energy for generation of (b3-1+Na)+ is decreased relative to that of (b3+17+Na)+ when the aromatic amino acid is located in a position such that it would lead to the formation of the highly conjugated oxazolinone, thus increasing the formation rate of the former species compared to the latter. 60 Summary The purpose of this study was to determine the influence of 4-Aminomethylbenzoic acid on the formation of the rival (b3-1+cat)+ and (b3+17+cat)+ species. Generation of these product ions involves cleavage of the same amide bond, but through entirely different mechanisms. For model peptides with general formula A(4Ambz)AX and A(4Ambz)GX, our study shows that formation of the (bn-1+cat)+ species is favored over the (bn+17+cat)+ species when the amide bond cleaved is one residue to the C-terminal side of the 4Ambz residue. We attribute this effect to the potential formation of a highly conjugated oxazolinone species as (b3-1+cat)+. Energy-resolved CID, which measures the collision energies necessary to generate product ions from a particular precursor ion, shows that formation of (b3-1+cat)+ occurs at significantly lower collision energies when the 4Ambz residue is present to affect the reaction pathway. This result is suggestive of an influence of the aromatic residue on the activation energy of the reaction to produce (b3-1+cat)+. Placing residues with bulky side groups at the C-terminus, which are known to enhance formation of (b3+17+cat)+, caused a modest increase in relative abundance of the product. However, generation of (b3-1+cat)+ remained the dominant dissociation pathway. 61 H N H2N O N H O H N H2N H N O H N N H H2N GGGA OH AAAG OH A(AMBz)AA OH A(AMBz)AV OH A(AMBz)AG OH A(AMBz)VG O O O H N OH O O N H O H N O O O O N H H N H2N H N O O O O N H H N H2N H N O O O O N H H N H2N H N O O O H2N H N O H N H2N O O N H H N O OH (AMBz)AAG O O N H H N AA(AMBz)G O OH O Figure 4.1 Sequence/structures of model peptides used to study the influence of 4AMBz on the formation of (b3-1+cat)+ and (b3+17+cat)+. 62 100 R. I. (%) 80 (a) y2 60 a4 + + 215 20 125 150 175 (b) 196 200 225 250 275 300 + (b3+17+Li) 60 40 + (b3-1+Li) 20 178 0 150 100 80 R. I. (%) + b4 /(M-H2O+H) + 172 40 80 175 200 (c) 225 212 250 275 250 275 + (b3+17+Na) 60 40 + (b3-1+Na) 20 194 0 150 100 80 R. I. (%) b3 147 0 100 100 R. I. (%) 243 + 175 (d) 200 225 + (b3-1+Ag) 278,280 60 + (b3+17+Ag) 40 20 0 200 296,298 + (a3-1+Ag) 250,252 225 250 275 300 325 350 375 m/z Figure 4.2 CID (MS/MS) spectra generated from cationized GGGA: (a) H+ cationized, (b) Li+ cationized, (c) Na+ cationized and (d) Ag+ cationized. 63 400 100 R. I. (%) 80 (a) b2 40 125 271 175 200 225 250 275 300 238 192 220 + (a3+17+Li) 60 + (b3+17+Li) + (b3-1+Li) 40 20 80 R. I. (%) 169 150 (b) 260 0 150 100 175 200 (c) 225 250 275 300 325 236 + (b3-1+Na) + (b3+17+Na) 254 60 + (a3-1+Na) 208 40 20 293 0 150 100 80 R. I. (%) + + 143 20 80 b3 147 60 0 100 100 R. I. (%) y2 214 + 175 200 225 (d) 250 + (b3-1+Ag) 275 300 325 350 320,322 60 40 + (a3-1+Ag) 20 0 200 292,294 220 240 260 280 300 320 340 360 380 400 420 m/z Figure 4.3 CID (MS/MS) spectra generated from cationized AAAG: (a) H+ cationized, (b) Li+ cationized, (c) Na+ cationized and (d) Ag+ cationized. 64 100 (a) R. I. (%) 80 40 20 R. I. (%) 80 250 275 + (b3-1+Li) 300 325 350 375 400 325 350 375 400 325 350 375 400 325 350 375 400 282 60 300 40 20 80 R. I. (%) 225 (b) 0 200 100 225 250 (c) 275 + (b3-1+Li) 300 282 300 60 40 20 0 200 100 80 R. I. (%) 276 60 0 200 100 225 250 275 (d) 300 + (b3-1+Na) 298 60 40 20 0 200 100 80 R. I. (%) + b3 225 250 275 300 (e) + (b3-1+Ag) 383 60 40 20 0 225 355 250 275 300 325 350 375 400 425 450 475 m/z Figure 4.4 CID (MS/MS) spectra generated from cationized A(4AMBz)AG: (a) H+ cationized, (b) Li+ cationized, (c) Li+ cationized, isolation and storage of (b3-1+Li)+, (d) Na+ cationized and (e) Ag+ cationized. 65 100 R. I. (%) 80 (a) + (b3-1+Na) 60 40 + (b3+17+Na) 20 0 200 100 R. I. (%) 80 225 250 275 300 325 375 400 350 375 400 350 375 400 350 375 400 + (b3-1+Na) 40 + (b3+17+Na) 20 316 225 250 (c) 275 300 325 284 + (b3-1+Na) 60 40 + (b3+17+Na) 20 302 0 200 100 80 350 298 60 80 R. I. (%) 316 (b) 0 200 100 R. I. (%) 298 225 250 (d) 275 300 325 284 + (b3-1+Na) 60 302 + (b3+17+Na) 40 20 0 200 225 250 275 300 325 m/z m/z Figure 4.5 CID (MS/MS) spectra generated Na+ cationized peptides: (a)A(4AMBz)AA, (b) A(4AMBz)AV, (c) A(4AMBz)GA and (d) A(4AMBz)GV. 66 100 (a) 316 + (b3+17+Na) R. I. (%) 80 60 40 + (M+Na) 373 -CO2 -H O, -H2O 2 329 NH3 355 338 20 0 200 100 225 250 275 300 325 350 375 400 298 (b) + (b3-1+Na) + (b3+17+Na) R. I. (%) 80 316 60 + (a3-1+Na) 270 40 + -H2O 355 20 0 200 225 250 275 300 325 350 (M+Na) 373 375 400 m/z Figure 4.6 CID (MS/MS) spectra generated Na+ cationized peptides: (a) AA(4AMBz)G, (b) (4AMBz)AAG. 67 O N H H N H+ H2 N O H N O OH O Proton transfer O N H H N H2 N H N H+ O OH O O O H N H+ O H N H 2N N+ H O O O N+ H H N H 2N O Scheme 9 O H2 N OH b3 + Reaction mechanism for formation of (b3)+ fromA(4AMBz)AG. 68 O OH 1.0 + (b3-1+Na) , AAGV + (b3+17+Na) , AAGV Fraction of ion abundance 0.8 + (b3-1+Na) , A(4AMBz)GV + (b3+17+Na) , A(4AMBz)GV 0.6 0.4 0.2 0.0 0.005 0.006 0.007 0.008 Applied CID voltage/DOF Figure 4.7 CID profiles for Na+ cationized AAGV and A(4AMBz)GV). 69 0.009 CHAPTER V Formation of (b3-1+cat)+ Ions from Metal-cationized Tetrapeptides Containing βAlanine, γ-Aminobutyric Acid or ε-Aminocaproic Acid Residues Introduction Formation of a 5-member cyclic intermediate is thought to be an integral part of the reactions to produce the bn+ and yn+ sequence ions from protonated peptides [19,20,23-26], and analogous (bn-1+cat)+ products from metal-cationized peptides through the “oxazolone” mechanism (shown for b3+ from AAAG in scheme 1a). The (bn+17+cat)+ species are prominent, if not the preferred, dissociation products for Li+ and Na+ cationized peptides [33,34,36,48]. The mechanism for the (bn+17+cat)+ pathways is also thought to involve cyclization and intramolecular nucleophilic attack with proton transfer (scheme 1b) [31,32,35,52]. The pathway depicted in scheme 1b includes concerted opening of the cyclic intermediate and elimination of CO and an imine. Gronert, Lebrilla and coworkers have proposed instead a plausible route that involves an anhydride intermediate after ring opening [52]. In any case, the salient feature is that, as in the case of the bn+/(bn-1+cat)+ pathway, the electrophilic site of attack in the (bn+17+cat)+ pathway is the carbonyl carbon of the amide group being cleaved. In this case, however, the nucleophile is the carbonyl oxygen of the C-terminal carboxylic acid. Recently we investigated and reported on the incorporation of “alternative” amino acids such as β-alanine (βA), γ-aminobutyric acid (γABu), ε-aminocaproic acid (Cap), and 4-aminomethylbenzoic acid (4AMBz) into the sequence of a model peptide, and the effect(s) of these residues on relative product ion intensities [29, 39]. The goal of this third study was to determine the effect on fragmentation patterns of changing the size of the putative cyclic intermediate formed during the nucleophilic attack. For protonated 70 peptides, the presence of βA, γAbu or Cap in XAAG, AXAG, and AAXG (where X represents the position of the “alternative” amino acid) inhibited or completely suppressed formation of specific bn+ and yn+ ions [39]. This observation was attributed to the prohibitive effect of forcing cyclization and intramolecular nucleophilic attack to progress through larger cyclic intermediates, which should mainly be kinetically slower to form and entropically less favored, when the larger amino acids were used. Cyclization is prohibited when the residue is 4AMBz because the rigid aromatic ring separates the nucleophile from the electrophilic site of attack. For metal (Li+, Na+ and Ag+) cationized peptides, similar suppression and inhibition of the formation of (b3+17+cat)+ was observed when the “alternative” amino acids were placed at the C-terminus of the model peptide acetyl-FGGX [29]. In this third study, the influence of βA, γAbu and Cap on the tendency to form (b3-1+cat)+ products from Li+, Na+ and Ag+ cationized AAXG was investigated. The initial hypothesis was that the potential prohibitively large cyclic intermediates would suppress formation of (b3-1+cat)+, as was observed for the protonated versions of the peptide. However, the metal cations may coordinate with the peptide through interactions with multiple amide carbonyl O atoms, and thus kinetically assist formation of a reactive configuration from which nulceophilic attack occurs. As we show here, (b3-1+cat)+ is a prominent, if not dominant, reaction product from the metal cationized AAXG. More importantly, isotope labeling demonstrates that formation of the product involves the transfer of an H atom that originates from an α-carbon position of the amino acid in position X. 71 Results CID of AA(γAbu)G The sequences and structures of the model peptides included in this study are shown in figure 5.1. Figure 5.2 shows CID (MS/MS stage) spectra for AA(γAbu)G cationized by: H+ (2a), Li+ (2b), Na+ (5.2c) and Ag+ (both the 107Ag+ and 109Ag+ isotopic peaks, (5.2d). For the protonated version of AA(γAbu)G, elimination of the full Cterminal G amino acid (75 mass units, u) to generate b3+ would furnish an ion peak at m/z 228. However, as in our earlier investigation [39], b3+ is not observed for AA(γAbu)G and the dominant product ion generated was instead y2+ at m/z 161. Suppressed formation of b3+ for the peptide containing γAbu is consistent with, at least in part, potential changes to the rates of the putative ring-closure step leading to nucleophilic attack in the “oxazolone” mechanism: the 7-member ring for γAbu should be kinetically slower to form than the conventional 5-member rings that are postulated as intermediates for αamino acids. For Li+ and Na+ cationized versions of AA(γAbu)G (figures 5.2b and c, respectively), the (b3+17+cat)+ product, generated by neutral loss of 57 mass units (u), was prominent. Other product ions observed included (M-H2O+cat)+, (y3-1+cat)+, (a3-1+cat)+ and (y2-1+cat)+. A more important observation, however, was the appearance of the (b3-1+cat)+ product. The relative abundance of (b3-1+cat)+ is comparable to that of (b3+17+cat)+ for the Li+ and Na+ cationized versions of the peptides, and is the dominant product ion generated from the Ag+ cationized version of the peptide. Thus, despite the potential prohibitive effect of the larger cyclic intermediates that presumably suppress formation of b3+ for the protonated peptides, the analogous (b3-1+cat)+ ion is prominent 72 in the spectra derived from the metal cationized peptides. Formation of (b3-1+cat)+ was not observed for the metal cationized versions of AA(4AMBz)G (spectra not shown), consistent with the hypothesis that prohibiting cyclization altogether blocks formation of the bn+/(bn-1+cat)+ products. CID of deuterium exchanged and labeled AAXG Figure 5.3 shows the CID spectra generated from (M+D)+ of AAXG peptides for which exchangeable H atoms were replaced with D by incubation of the peptides in a mixture of D2O and CH3OD. For D+-cationized AAAG (figure 5.3a), the b3+ ion appears as multiple isotopic peaks. Isolation and storage of the m/z 218 isotopic peak, without imposed collisional activation (spectra not shown) suggested that the m/z 217, 216 and 215 isotopic peaks are instead generated by D for H back-exchange through collisions with adventitious H2O in the ion trap. This was confirmed by monitoring exchange of D for H with increasing isolation times. Investigation of the cause for the rapid D for H exchange for b3+ derived from AAAG is beyond the scope of this report, and is being studied in greater detail using a more extensive set of model peptides. As shown in figure 5.3b, the b3+ ion at m/z of 218, via loss of 78 u, was also observed following CID of deuterium exchanged AA(βA)G, consistent with loss of fully deuterium exchanged glycine from the C-terminus. D for H back exchange was minimal, and became significant only at isolation times longer than 1 second (data not shown). For AA(βA)G, AA(γAbu)G and AA(Cap)G (figures 5.3b through d, respectively), formation of y2+ following CID of (M+D)+ occurred through the net elimination of 144, consistent with loss of the two N-terminal A residues, each with a single D atom. Isolation and 73 storage of y2+, without imposed collisional activation, showed that the tendency for D for H back exchange was negligible. The CID spectra generated from Li+ and Na+ cationized versions of the deuterium-exchanged forms of AAXG were qualitatively similar. For the sake of brevity, the results from the Li+ versions are presented here as representative of both systems. As shown in figure 5.4 for Li+ cationized, deuterium-exchanged AAAG, the (b3+17+Li)+ and (b3-1+Li)+ products were observed at m/z 243 and 223, respectively. These m/z values correspond to product ions generated by elimination of 58 and 78 u, respectively. The formation pathways proposed for both the (bn-1+cat)+ and (bn+17+cat)+ products involve transfer of H atoms from exchangeable sites, specifically amide and acid positions for the former and latter, respectively. The elimination of 58 and 78 u during formation of (b3+17+Li)+ and (b3-1+Li)+ from deuterium-exchanged AAAG is therefore consistent with the respective proposed mechanistic pathways, and demonstrates that generation of the products does not include significant scrambling of isotope labels or transfer of H atoms from positions other than the amino terminus, amide positions, or the C-terminal acid group. Both the (b3+17+Li)+ and (b3-1+Li)+ were isolated and stored in the ion trap, without imposed collisional activation, for periods ranging from 10 msec to 1 sec (spectra not shown): no appreciable D for H back exchange for the ions was observed. For Li+ cationized, deuterium-exchanged AA(βA)G (5.4b), AA(γAbu)G (5.4c) and AA(Cap)G (5.4d), formation of (b3+17+Li)+ continued to involve elimination of 58 u, consistent with transfer of the C-terminal acid-position D atom during the dissociation reaction (as shown with a C-terminal H atom in scheme 1b). However, the (b3-1+Li)+ ion appeared as two isotopic peaks (m/z 223, 224; 237, 238 and 265, 266 for X = βA, γAbu 74 and Cap, respectively) that arise through neutral losses of 77 or 78 u. Elimination of 78 u is consistent with transfer of an amide-position D atom to the C-terminal residue, and loss of fully deuterium exchanged glycine to furnish (b3-1+Li)+. The neutral loss of 77 u is noteworthy, because it signals transfer to the C-terminal G residue of an H atom, rather than a D atom, in the dissociation pathway. Because the peptides were fully D exchanged (amino, amide and acid positions labeled with D), the transfer of an H atom implicates one of the methylene groups within the residue at position X. As is evident in the comparison of the spectra in figure 5.4, the tendency to lose 77 u rather than 78 u increases following the trend βA < γAbu < Cap, i.e, as the size of the amino acid at which cyclization and intramolecular nucleophilic attack should occur to produce (b3-1+cat)+ increases. Similar trends were observed for the Na+ cationized versions of the deuteriumexchanged peptides. As in the case of the AAAG peptide, both the (b3+17+Li)+ and (b3-1+Li)+ derived from Li+ and Na+ cationized AA(βA)G, AA(γAbu)G, and AA(Cap)G were isolated and stored in the ion trap, without imposed collisional activation, for periods ranging from 10 msec to 1 sec (spectra not shown). No appreciable D for H back exchange for the respective ions was observed. The (a3-1+Li)+ ion at m/z 195 was a prominent product in the spectrum derived from Li+ cationized, fully deuterium exchanged AAAG. CID (MS3 stage, spectrum not shown) of (b3-1+Li)+ from the deuterium exchanged AAAG led primarily to (a3-1+Li)+ via elimination of CO. The (a3-1+Li)+ product is absent in the CID spectra generated from Li+ cationized, fully deuterium exchanged AA(βA)G (5.4b), AA(γAbu)G (5.4c) and AA(Cap)G (5.4d), nor was the ion generated by CID of (b3-1+Li)+ derived from the respective peptides, as discussed in more detail in a later section. 75 Figure 5.5 shows the CID spectra for Ag+ cationized AAAG (5.5a), AA(βA)G (5.5b), AA(γAbu)G (5.5c) and AA(Cap)G (5.5d). For the sake of clarity, the spectra shown in figure 5.5 were generated using CID of only the 109Ag+ adduct of the peptides. The (b3-1+109Ag)+ ion is the dominant product generated from each of the peptides, and (b3+17+109Ag)+ appeared at relative intensities < 5% for the AA(βA)G, AA(γAbu)G and AA(Cap)G peptides. As in the case of the Li+ and Na+ cationized versions, formation of (b3-1+109Ag)+ generated from deuterium-exchanged AAAG involved primarily elimination of 78 u, which represents fully deuterium exchanged glycine. However, loss of 77 u was observed from the deuterium-exchanged AA(βA)G, AA(γAbu)G and AA(Cap)G, and the fraction of product ion derived from this pathway increased systematically with the size of the “alternative” amino acid. No D for H back exchange was observed when either (b3+17+109Ag)+ or (b3-1+109Ag)+ derived from Ag+ cationized AA(βA)G, AA(γAbu)G, and AA(Cap)G were isolated and stored in the ion trap, without imposed collisional activation, for periods ranging from 10 msec to 1 sec (spectra not shown). As in the case of the Li+ (and Na+) cationized versions of deuterium exchanged AA(βA)G, AA(γAbu)G and AA(Cap)G, the (a3-1+109Ag)+ product was not generated by CID of (b3-1+109Ag)+. The CID spectra shown in figures 5.4 and 5.5 demonstrate that formation of (b3-1+cat)+ from metal cationized AA(βA)G, AA(γAbu)G and AA(Cap)G involves, in part, transfer of an H atom that does not originate in an exchangeable position. To determine the specific position from which the H atom is transferred during formation of (b3-1+cat)+, the CID of metal cationized AA(α−d2-γAbu)G was examined. This peptide was chosen for study because α-d2 labeled γAbu is commercially available, and because 76 our initial hypothesis was that the α-C position is the most likely origin of the H atom. In figures 5.6 and 5.7, high-resolution ZoomScan spectra collected after CID of native AA(γAbu)G and AA(α−d2-γAbu)G are compared for the Li+ and 109Ag+ cationized forms of the peptides, respectively. For AA(α−d2-γAbu)G (figures 5.6b and 5.7b), the (b3-1+cat)+ ion appears as two isotopic peaks, which are shifted higher by 1 and 2 mass units compared to the unlabeled version of the peptide. The higher mass isotopic peaks (m/z 236 and 338 for Li+ and Ag+, respectively) are generated by the net elimination of 75 u and formation of (b3-1+cat)+ via a reaction that involves transfer of an H atom from an amide position to the departing, C-terminal G residue. The lower mass peaks (m/z 235 and m/z 337 for the Li+ and Ag+ cationized versions, respectively) correspond to formation of (b3-1+cat)+ by transfer of a D atom, thus supporting the hypothesis that the α-C position is the source of the non-exchangeable H atom in the reaction pathway. Regardless of the metal cation, exclusive loss of 78 u was observed following CID of AA(α−d2-γAbu)G that was incubated in D2O/CH3OD to induce H/D exchange (spectra not shown). This result suggests that only H (or D) atoms from amide N positions or the α-carbon position of residue X are transferred in the reaction to produce (b3-1+cat)+. Proposed mechanism for (b3-1+cat)+ with transfer of α-position H atom The results shown thus far demonstrate that placement of larger amino acids such as βA, γAbu or Cap in position X of AAXG does not appreciably inhibit formation of (b3-1+cat)+ from the model peptide, unlike the results obtained for b3+ from protonated versions of the same peptides [39]. The pronounced transfer of an H atom from an αcarbon position suggests that formation of (b3-1+cat)+ occurs, at least in part, through a 77 mechanistic pathway distinct from that proposed for peptides with conventional α-amino acids. Two reasonable general pathways can be envisioned for (b3-1+cat)+ from AAXG where X= βA, γAbu or Cap: one involving formation of cyclic intermediate larger than the 5-member ring proposed for α-amino acids, and a second pathway that instead involves tautomerization and generation of a metal-cationized ketene. Using the peptide AA(γAbu)G for discussion, a mechanism involving intramolecular nucleophilic attack and formation of a cyclic intermediate is shown in scheme 10. Once the cyclic intermediate is formed, proton transfer from the amide N position of the γAbu residue to the amide N atom of the C-terminal glycine residue must occur. Cleavage of the C-terminal amide bond would then liberate glycine as a neutral and furnish 3H-tetrahydro-1,3-oxazepine, a 7-membered ring analogue to the oxazolinone (a 5-membered ring) generated by α-amino acids. In our earlier study of protonated peptides, residues such as γAbu and Cap were found to suppress formation of bn+ ions when the residues were positioned such that they influence formation of the cyclic intermediate. The larger cyclic intermediates were thought to be kinetically and entropically less favored, thus leading to diminished ion yields. In the present case, formation of (b3-1+cat)+ for which amide position H (or D) atoms are transferred to the departing neutral by the mechanism shown in scheme 10 suggests that the metal ion may facilitate nucleophilic attack by coordinating with the proper amide carbonyl oxygen atoms to assist formation of the reactive configuration, thus kinetically assisting the reaction pathway. 78 Though the reaction shown in scheme 10 may account for generation of (b3-1+cat)+ with specific transfer of an amide-position H (or D) atom, it cannot explain the formation of the same species when an H atom from an α-carbon position is transferred. Assuming formation of the same intermediate after nucleophilic attack, transfer of the α-position D atom to the O atom would have to be accompanied by opening of the ring as shown in scheme 11. However, the cyclic intermediate depicted in scheme 11 has no properly located keto group to participate in keto-enol tautomerization, or otherwise render the α-deuterium atoms sufficiently acid; so it is difficult to mechanistically explain the transfer of deuterium from the α-carbon position necessary to complete the reaction to produce (b3-1+cat)+ and account for the splitting of the product ion as observed in figures 5.6 and 5.7 A rational alternative to a mechanism that involves cyclization is formation of a metal-cationized ketene as shown in scheme 12. The appearance of (b3-1+cat)+ in the CID spectra of metal-cationized AAXG, when X = γAbu or Cap, but not b3+ from protonated analogues, and that generation of (b3-1+cat)+ from AAXG involves transfer of an H atom from the α-position of X suggests that the acidity of this H atom has been enhanced by the intervention of the metal ion. The normal pKa of this H atom is about 25-30. However, as suggested below, this H atom acquires the character of allylic to an iminium ion prior to fragmentation. The pKa of H in an allylic position to electronegatively-substituted imines such as oxime tosylates cannot be much higher than 16-18 because its removal by a base such as ethoxide (-OEt) promotes the Neber rearrangement via an azirine [62]. Moreover, condensation of a CH3 group, adjacent to an iminium ion, with orthoformate to form a cyanine dye in acetate buffer [63] indicates that 79 its pKa must be significantly lower than 16-18. Any process that interferes with the production of carbocation A’ (scheme 12) would disfavor loss of glycine. Such a process is cyclization involving nucleophilic attack by the oxygen of the penultimate alanine residue of AAXG (iminium ion A, scheme 11). When X = Cap, this ring is ninemembered, whereas X = γAbu, βA or Ala leads to seven-, six- and five-membered rings, respectively. It is well-known that formation of medium-sized rings (sizes 7-9) is least favored based on both thermodynamic and kinetic factors. Thus, the acyl ion condensation affords these rings in lower yields than both bigger or somewhat smaller ones [64], and the Dieckmann condensation yields virtually no product corresponding to medium-sized rings [65]. Therefore, we expect the loss of C-terminal glycine to be greatest with X = Cap because of least competition with formation of A’, and diminish in the order γAbu > βA > Ala. If the (b3-1+cat)+ ion is indeed a metal-cationized ketene one would not expect it to lose CO upon further dissociation. Ketenes (as odd-electron species) by themselves do lose CO upon electron impact to produce electron deficient carbenes. However, formation of such carbenes in the present case would be discouraged by the proximity of electron deficient metal ions. Figure 5.8 shows spectra generated by CID (MS3 stage) of (b3-1+Li)+ (5.8a) and (b3-1+109Ag)+ (5.8b) derived from the respective metal-cationized versions of AA(γAbu)G. For neither species is the loss of CO a prominent pathway. The dominant product fragmentation pathway for (b3-1+Li)+ involves either elimination of 18 u, presumably as H2O, or loss of 44 u. Elimination of 44 u was observed by our group for dissociation of (b3-1+cat)+ ions generated from metal-cationized AXGG, where X=βA, γAbu or Cap, and attributed to elimination of CO2 to furnish a metal-cationized azirine 80 [66]. In the prior study, the (b3-1+cat)+ ions were all 5-member ring oxazolinones, and decomposition of the azirine led to formation of a metal-cationized nitrile product. What specific atoms make up the neutral(s) eliminated at 44 u in the present study is not known and will be determined using a more extensive series of isotope labeled peptides. Other dissociation pathways observed were formation of (b2-1+Li)+ and (a2-1+Li)+ via elimination of 85 u (residue mass of γAbu) and 113 u (residue mass of γAbu and CO), respectively. For (b3-1+109Ag)+ , similar dissociation pathways were observed. However, formation of (b2-1+109Ag)+ and (a2-1+109Ag)+ was more favored compared to loss of H2O or 44 u. It is clear from the spectra in figure 5.8, and the lack of (a3-1+cat)+ products in the CID spectra of Li+ and Ag+ cationized AA(γAbu)G and AA(Cap)G (figures 5.3 and 5.4), that (b3-1+cat)+ with the larger “alternative” amino acids does not dissociate via the elimination of CO as do conventional bn+/(bn-1+cat)+ ions. Loss of CO from the (bn-1+cat)+ and analogous bn+ species generated from AAAG, and related peptides composed of α-amino acids, is assisted by concomitant facile formation of an imine product (scheme 1a). For AA(βA)G, AA(γAbu)G and AA(Cap)G, cyclic (b3-1+cat)+ products formed via the pathway shown in scheme 10 would not be expected to form the associated (a3-1+cat)+ species because opening of the ring, with concomitant elimination of CO, can not lead to formation of an imine product as depicted in scheme 1 without significant rearrangement. As noted above, metallated ketenes likely also would not eliminate CO. Loss of H2O from (b3-1+Li)+ and (b3-1+Na)+ probably occurs through a retroKoch or retro-Ritter type pathway and elimination of an amide O atom, as proposed first 81 by Reid and coworkers [55] for protonated peptides and supported by subsequent experiments by our group with metal-cationized peptides [28, 35, 56]. The formation of (b2-1+cat)+ and (a2-1+cat)+ from (b3-1+cat)+ can instead be attributed to the conventional “oxazolone” pathway, with attack by the carbonyl O atom of the alanine residue adjacent to the N-terminus upon either a C atom within the large ring formed in scheme 10, or the amide group between alanine and the γAbu-ketene to the peptide generated in scheme 12. Regardless of the precursor ion structure, such a nucleophilic attack would furnish a metal-cationized oxazolinone as (b2-1+cat)+ which could decompose further, through loss of CO, to produce (a2-1+cat)+. CID (MS4 stage, spectra not shown) of (b2-1+Li)+ and (b2-1+Ag)+ derived from AA(γAbu)G or AA(Cap)G produces the (a2-1+cat)+ species via the loss of CO. Apparent influence of X on yield of other product ions Another interesting observation was the apparent influence of X in AAXG on the relative abundance of the (M-H2O+cat)+ and (b3+17+cat)+ products relative to (b3-1+cat)+. As noted above, an investigation of the elimination of H2O from model, metal cationized peptide-esters showed that the preferred pathway likely involves a retroKoch type-process that involve loss of an amide-position carbonyl O atom [32, 33]. This is opposed to loss of the C-terminal -OH group through a pathway similar to that operative for generation of the (bn-1+cat)+ and b3+ ions shown in scheme 1a. In the previous study, loss of H2O was shown to involve loss of both amide N position and αcarbon position H atoms [56], consistent in the present study with the significant fraction of the (M-H2O+Li)+ generated from the fully deuterium exchanged form of AA(βA)G 82 and AA(γAbu)G via the loss of HDO rather than D2O. The reason for the enhanced loss of H2O from AA(βA)G and AA(γAbu)G is not clear, but we have found that the intensity of (M-H2O+cat)+ increases when the favored (bn+17+cat)+ pathway is inhibited or suppressed [29]. Noting the fact that the (b3-1+Li)+ species appears as two isotopic peaks, the ratio of the abundance of (b3-1+Li)+ relative to (b3+17+Li)+ increases from ca. 0.75 (measured using integrated peak areas) for AAAG to ca. 1.5 for AA(βA)G and AA(γAbu)G, and further to nearly 5 for AA(Cap)G. The change in relative abundance is surprising given the fact that the (b3+17+Li)+ is often the preferred, if not dominant, product generated from metal-cationized peptides. We recently investigated the competition between formation of (b3-1+Li)+ and (b3+17+Li)+ from A(4AMBz)AG and found that there was a significant increase in the intensity of (b3-1+Li)+ for the peptides that contain the 4AMBz residue, and in some cases the complete elimination of the (b3+17+Li)+ pathway [67]. The influence of the 4AMBz residue was attributed to the fact that (b3-1+Li)+ would be a highly-conjugated species containing an aromatic ring substituent. Comparison of CID profiles suggested that the competition may be influenced by a decrease of the critical energy for generation of (b3-1+cat)+ relative to that of (b3+17+cat)+. However, in the present study the initial assumption was that the larger amino acids would suppress formation of (b3-1+cat)+ because of the larger cyclic intermediates, and in doing so presumably increase the competitiveness of the (b3+17+cat)+ product. As shown in scheme 1b, formation of (b3+17+cat)+ requires attack on an amide C atom by the C-terminal carboxyl group to generate an oxazolidinone intermediate, whereas formation of (b3-1+cat)+ via the ketene pathway would involve attack by the adjacent 83 nitrogen atom on the same carbocation (scheme 12), to produce an iminium ion. Preferred formation of (b3-1+cat)+ over (b3+17+cat)+ as the size of the amino acid in position X increases may be rationalized by considering that the nitrogen atom may be more nucleophilic than the carbonyl group that would attack in the pathway shown in scheme 1b, and thus make formation of the former product more favorable. The (y3-1+cat)+ ion was also more prominent in the CID spectra generated from Li+ and Na+ cationized AA(γAbu)G and AA(Cap)G when compared to AAAG. Computational studies by Paizs and Suhai provide compelling evidence for an integrated pathway that involves simultaneous cleavage of the C-C bond of the N-terminal amino acid residue, and the N-terminal amide C-N bond to generate y3+, a1+ and CO [68]. Such a pathway was found, for protonated glycine-glycine, to be energetically more favorable than one involving formation of an aziridinone product via cyclization and attack by the N-terminal amino group. In the present study, it is not clear what role the metal ion may play in enhancing the formation of (y3-1+cat)+, and a more detailed investigation of the enhanced formation of the species is currently underway using a larger group of model peptides. 84 Summary In this study we examined the effect of β-alanine, γ-aminobutyric acid or εaminocaproic acid residue on the formation of (b3-1+Cat)+ products from metal(Li+, Na+ and Ag+) cationized peptides. Although, the “alternative” amino acids suppress the formation of b3+ from protonated peptides with the sequence AAXG due to the prohibitively large cyclic intermediate in the “oxazolone” pathway, the results above show that abundant (b3-1+Cat)+ are generated from metal cationized versions of AAXG. Using a group of D-labeled and exchanged peptides, we found that the formation of (b3-1+cat)+ involves transfer of either amide or α-carbon position H atoms. An important observation was that the tendency to transfer the α-carbon position H atoms increase with the size of the alternative amino acid in position X. 85 A H N H2N O N H O N H H N H2 N H N O Figure 5.1 O H N O H N O OH O γAbu O O H N N H O H2 N OH βA O H2N O H N OH O Cap O H N N H O OH O Sequence/structures of model peptides used to study the influence of βA, γAbu and Cap on formation of (b3-1+cat)+ from metal-cationized AAXG. 86 R. I. (%) 100 161 a 80 + y2 60 + b3 40 + y3 144 20 232 0 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 R. I. (%) 100 80 + [b3-1+Li] b + [b3+17+Li] 234 60 274 252 + 40 [y3-1+Li] + [y2-1+Li] 20 238 167 0 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 R. I. (%) 100 80 + [b3-1+Na] c [y3-1+Na] 60 + 40 [y2-1+Na] 20 [a3-1+Na] 250 268 + [b3+17+Na] + 290 + 254 222 183 0 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 R. I. (%) 100 338 d 80 336 + [b3-1+Ag] 60 [b3+17+Ag] + 40 [y2-1+Ag] 20 267 269 352 + 354 0 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 m/z Figure 5.2. CID (MS/MS) spectra generated from AA(γAbu)G series cationized by: (a) H+, (b) Li+, (c) Na+ and (d) 107Ag+ and 109Ag+. 87 R. I. (%) 100 a 152 + y2 + b2 80 60 146 40 217 + b3 20 218 215 a3* 151 216 170,171 0 140 R. I. (%) 100 80 b 150 160 170 180 190 200 210 220 230 152 60 40 20 218 0 140 R. I. (%) 100 80 c 150 160 170 180 190 200 210 220 230 166 60 40 20 0 150 R. I. (%) 100 80 d 160 170 180 190 200 210 220 230 240 194 60 40 20 0 180 190 200 210 220 230 240 250 260 270 m/z Figure 5.3. Spectra generated by CID (MS/MS) of D+ cationized, fully deuteriumexchanged: (a) AAAG, (b) AA(βA)G, (c) AA(γAbu)G and (d) AA(Cap)G. 88 R. I. (%) 100 a 80 + + [b3+17+Li] 223 [a3-1+Li] 60 243 + [b3-1+Li] 195 + [y3-1+Li] 40 20 229 0 180 R. I. (%) 100 80 190 200 210 220 230 b 240 250 243 270 263 262 223 60 260 224 40 229 20 0 180 R. I. (%) 100 80 190 200 210 220 230 240 250 260 257 c 270 275 276 238 237 60 243 40 20 0 190 R. I. (%) 100 80 200 210 220 230 240 250 260 270 280 266 d 60 40 265 303 304 285 271 20 0 220 230 240 250 260 270 280 290 300 310 m/z Figure 5.4. Spectra generated by CID (MS/MS) of Li+ cationized, fully deuterium-exchanged: (a) AAAG, (b) AA(βA)G, (c) AA(γAbu)G and (d) AA(Cap)G 89 R. I. (%) 100 a 80 325 + [b3-1+Ag] 60 + [a3-1+Ag] 40 326 297 20 0 290 R. I. (%) 100 300 310 320 b 80 330 340 350 360 350 360 325 60 326 40 20 345 0 290 R. I. (%) 100 300 310 320 c 80 330 339 340 340 60 40 359 20 0 300 R. I. (%) 100 80 310 320 330 340 d 350 360 370 368 60 367 40 20 387 0 330 340 350 360 370 380 390 400 m/z Figure 5.5. Spectra generated by CID (MS/MS) of 109Ag+ cationized, fully deuterium-exchanged: (a) AAAG, (b) AA(βA)G, (c) AA(γAbu)G and (d) AA(Cap)G. 90 100 (a) 234 (b3-1+Li)+ 80 R. I. (%) (b3+17+Li)+ 252 60 (y3-1+Li)+ 40 238 20 0 230 100 234 236 238 240 242 244 246 (b) 80 R. I. (%) 232 60 248 250 252 254 (b3+17+Li)+ 254 (b3-1+Li)+ 235 236 (y3-1+Li)+ 40 240 20 0 232 234 236 238 240 242 244 246 248 250 252 m/z Figure 5.6 CID (MS/MS) spectra of Li+ cationized (a) AA(γAbu)G and (b) AA(α-d2-γAbu)G. 91 254 256 100 (a) (b3-1+Ag)+ 80 R. I. (%) 336 60 40 (b3+17+Ag)+ 354 20 0 332 100 336 (b) 80 R. I. (%) 334 337 338 340 342 344 346 348 350 352 354 356 338 (b3-1+Ag)+ (b3+17+Ag)+ 60 356 40 20 0 334 336 338 340 342 344 346 348 350 352 354 m/z Figure 5.7. CID (MS/MS) spectra of Ag+ cationized (a) AA(γAbu)G and (b)AA(α-d2-γAbu)G. 92 356 358 100 216 (a) 190 80 R. I. (%) -H2O -44 60 -71 40 -113 121 20 234 149 0 100 -85 (b3-1+Li)+ 163 125 150 175 200 225 250 251 (b) -85 R. I. (%) 80 -113 223 60 -43 40 293 -H2O 336 318 20 0 (b3-1+Ag)+ 225 250 275 300 325 350 m/z Figure 5.8. CID (MS3) spectra of (a) (b3-1+Li)+Ag+ and (b) (b3-1+Ag)+ derived from AA(γAbu)G. 93 AA(α-d2-γAbu)G+cat+ H N H2N O D N H O D H N O cat+ OH O H2N H N + D D NH H O : NH cat+ :O: N O H O H2N cat+ : - O H :O: N O H + D N D NH O O NH O N N :O: O H + N H OH D D H2N O H2N O OH (b3-1+cat)+ NH O OH N Scheme 10 cat+ O N H D D O cat+ : O OH OH H2N H2N O O cat+ D D Pathway to (b3-1+cat)+ from metal cationized AA(α-d2-γAbu)G through 7-membered cyclic intermediate, with transfer of amide position H atom 94 AA(α-d2-γAbu)G+cat+ O H N H2N D N H O D cat+ O H N OH O H2N : cat+ :O: NH N O H H2N X D O NH O O O H N O N H + cat+ OH D H N H O Scheme 11. N D O D N H H N+ D O OH :O: cat+ OH D N H O - : O O H2N OH D D H N H2N H N A O cat+ C O (b3-1+cat)+ Potential pathway to (b3-1+cat)+ from metal cationized AA(α-d2γAbu)G through 7-membered cyclic intermediate, with transfer of αcarbon position H atom. 95 AA(α-d2-γAbu)G+cat+ O H N H2N N H O : A’ :O: O H N - D N H H N+ O OH D :O: - : O cat+ O H N H2N OH D cat+ H2N O H + N : O OH O D N H cat+ O H N D O H N H2N D D N H O OH :O: - : O H N+ D cat+ O H B H N H2N O D N H O OH N D cat+ C O (b3-1+cat)+ Scheme 12. Pathway to (b3-1+cat)+ from metal cationized AA(α-d2-γAbu)G through alternative ketene mechanism. 96 CHAPTER VI CONCLUSIONS The objective of the first set of experiments in this study was to investigate the CID patterns for thallium(I) cationized peptides and compare them to those from Ag, Na and protonated versions. In comparison to H+, Na+, and Ag+, we found that lower desolvation temperatures and milder ionization conditions were required to produce abundant (M+Tl)+ ions, an observation that suggested weak binding of Tl+ by the entire suite of peptides. This conclusion was supported by appearance of prominent Tl+ signals in the CID spectra of each peptide. In addition, the activation amplitudes required to induce fragmentation (ca. 20-25%, 25-30% and 30-35% for Tl, Ag, and Na respectively) of Tl cationized peptides were comparable to those for Ag cationized versions and lower than Na cationized analogues. As a result of tendency to eliminate Tl+ during CID, use of MS/MS experiments to determine sequence from the C-terminus was not possible. The objectives of the second set of experiments was to investigate the influence of 4-Aminomethylbenzoic acid on the formation of the rival (b3-1+cat)+ and (b3+17+cat)+ species. Generation of these product ions involves cleavage of the same amide bond, but through entirely different mechanisms. For model peptides with general formula A(4Ambz)AX and A(4Ambz)GX, our study shows that formation of the (bn-1+cat)+ species is favored over the (bn+17+cat)+ species when the amide bond cleaved is one residue to the C-terminal side of the 4Ambz residue. As proposed in our earlier study of the effect of “alternative” amino acids on the CID of protonated peptides, it is likely that the influence of the 4AMBz residue observed here with the metal cationized peptides can 97 be attributed to generation of a highly-conjugated oxazolinone species as (b3-1+cat)+, which would increase the stability of this product relative to the rival (b3+17+cat)+ ion. Energy-resolved CID, which measures the collision energies necessary to generate product ions from a particular precursor ion, shows that formation of (b3-1+cat)+ occurs at significantly lower collision energies when the 4Ambz residue is present to affect the reaction pathway. This result is suggestive of an influence of the aromatic residue on the activation energy of the reaction to produce (b3-1+cat)+. Placing residues with bulky side groups at the C-terminus, which are known to enhance formation of (b3+17+cat)+, caused a modest increase in relative abundance of the product. However, generation of (b3-1+cat)+ remained the dominant dissociation pathway. The relative energies of precursor and product ions, and of transition states and relevant intermediates have yet to be determined, and would benefit from a comprehensive computational study. In the last set of experiments, the effect of the “alternative” βA, γAbu and Cap residues on the tendency to form (b3-1+cat)+ ion from metal cationized peptides was examined. For protonated versions of these peptides, the larger amino acids suppress formation of b3+ from peptides with general sequence AAXG, presumably due to the prohibitive effect of larger cyclic intermediates in the “oxazolone” pathway. However, we found that abundant (b3-1+cat)+ products are generated from Li+, Na+ and Ag+ cationized versions of AAXG, despite the potential negative effects of the large cyclic intermediates. Using a group of D-labeled peptides, we found that formation of (b3-1+cat)+ from the AAXG peptides involves transfer of both amide position H atoms, as well as those attached to C atoms. The most likely source of the H atom transferred is identified as the α-carbon position using AA(γAbu)G for which the α-C position of the 98 γAbu residue was labeled with D. 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