THE INFLUENCE OF CATION AND ALTERNATIVE AMINO ACIDS ON THE FRAGMENTATION PATHWAYS OF METAL CATIONIZED AND PROTONATED PEPTIDES. A Thesis by SILA OCHOLA

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1 TE IFLUECE F CATI AD ALTERATIVE AMI ACIDS TE FRAGMETATI PATWAYS F METAL CATIIZED AD PRTATED PEPTIDES A Thesis by SILA CLA B.ED, Egerton University, airobi, Kenya, 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 28

2 Copyright 28 by Sila duor chola All Rights Reserved

3 TE IFLUECE F CATI AD ALTERATIVE AMI ACIDS TE FRAGMETATI PATWAYS F METAL CATIIZED AD PRTATED 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

4 ACKWLEDGEMET 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. e 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 ational Science Foundation (CAREER-2398) and a Faculty Scholar Award from the Kansas Biomedical Research Infrastructure etwork. Funds for the purchase of the ESIinstrumentation were provided by the Kansas SF-EPSCoR Program and Wichita State University. iv

5 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 +, a + and silver cationized peptides, particularly with respect to the preferred product ions. For example, the former produce primarily (b n +17+Li) + while the latter preferentially generates (b n -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, a, 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 (b n +17+Tl) + or (b n -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 4- aminomethylbenzoic acid (4AMBz) residue on the relative intensities of (b 3-1+cat) + and (b cat) + fragment ions using tetrapeptides of the general formula A(4AMBz)AX and A(4AMBz)GX (where X = G, A, V). For Li + and a + cationized versions of the peptides there was a significant increase in the intensity of (b 3-1+cat) + for the peptides that contain the 4AMBz residue, and in some cases the complete elimination of the (b cat) + pathway. The influence of the 4AMBz residue is attributed to generation of a highly-conjugated oxazolinone species as (b 3-1+cat) +, which increases the stability of this product relative to the rival (b cat) + ion. This conclusion is supported by dissociation profiles, which suggest v

6 that the energetic requirements for generation of (b 3-1+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 (b 3-1+cat) + products from metal (Li +, a + and Ag + ) cationized peptides. The larger amino acids suppress formation of b + 3 from protonated peptides with general sequence AAXG (where X=β-alanine, γ-aminobutyric acid or ε- aminocaproic acid), presumably due to the prohibitive effect of larger cyclic intermediates in the oxazolone pathway. owever, abundant (b 3-1+cat) + products are generated from metal cationized versions of AAXG. Using a group of deuterium-labeled and exchanged peptides, we found that formation of (b 3-1+cat) + involves transfer of either amide or α-carbon position 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 atom, a mechanism involving formation of a ketene product as (b 3-1+cat) + is proposed. vi

7 TABLE F CTETS CAPTER PAGE CAPTER I CAPTER II ITRDUCTI...1 EXPERIMETAL METDS Mass Spectrometry.12 Electrospray Ionization..14 Quadrupole Ion trap Analyzer...17 Tandem Mass Spectrometry..2 Procedure...24 CAPTER III CAPTER IV CAPTER V Collision Induced Dissociation of Tl + -cationized peptides...28 Influence of a 4-aminomethylbenzoic acid residue on the competitive fragmentation pathways during CID of metal cationized peptides.48 Formation of (b 3-1+cat) + Ions from Metal-cationized Tetrapeptides containing β-alanine, γ-aminobutyric Acid or ε-aminocaproic Acid Residues 7 CAPTER VI REFERECES Conclusion vii

8 LIST F FIGURES FIGURE Figure 1.1 Figure 1.2 Figure 1.3 Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Figure 2.6 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 Figure 3.7 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 PAGE Biemann nomenclature for fragment ions b n and y n ions...3 Representative structures of protonated b and y ions 3 CID spectra of protonated AAXG.11 The components of mass spectrometer.13 Principle of electrospray ionization...16 Basic components of quadrupole ion trap analyzer...19 Collision induced dissociation of precursor ions...22 Principle of Tandem Mass Spectrometry...23 Multiple reaction vessel peptide synthesis apparatus 27 CID spectra of protonated and metal cationized VAAF 35 CID spectra of protonated and metal cationized YGGFL.36 CID spectra of protonated and metal cationized VGVAPG..37 Multi-stage CID of (LGGFL + Tl) + product ions..4 Multi-stage CID of (FGGLL + Tl) + product ions..41 Multi-stage CID of (FGGGL + Tl) + product ions..43 CID of (AGGFL + Tl) + product ions.44 Structures of the group of peptides analysed.62 CID spectra of protonated and metal cationized GGGA..63 CID spectra of protonated and metal cationized AAAG...64 CID spectra of protonated and metal cationized A(4AMBz)AG...65 viii

9 LIST F FIGURES (CTIUED) FIGURE Figure 4.5 Figure 4.6 Figure 4.7 Figure 5.1 Figure 5.2 Figure 5.3 Figure 5.4 Figure 5.5 Figure 5.6 Figure 5.7 Figure 5.8 PAGE CID spectra of a + cationized A(4AMBz)AX and A(4AMBz)GX..66 CID spectra of a + cationized AA(4AMBz)G and (4AMBz)AAG..67 CID profile of a + cationized AXGV (X= A or 4AMBz).69 Sequence/structures of model peptides used to study the influence of βa, γabu and Cap on formation of (b 3-1+cat) + from metal-cationized AAXG 86 CID (MS/MS) spectra generated from AA(γAbu)G series cationized by: (a) +, (b) Li +, (c) a + and (d) 17 Ag + and 19 Ag +.87 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 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 Spectra generated by CID (MS/MS) of 19 Ag + cationized, fully deuteriumexchanged: (a) AAAG, (b) AA(βA)G, (c) AA(γAbu)G and (d) AA(Cap)G 9 CID (MS/MS) spectra of Li + cationized (a) AA(γAbu)G and (b)aa(α-d 2 -γabu)g..91 CID (MS/MS) spectra of Ag + cationized (a) AA(γAbu)G and (b) AA(α-d 2 -γabu)g.92 CID (MS 3 ) spectra of (a) (b 3-1+Li)+Ag + and (b) (b 3-1+Ag) + derived from AA(γAbu)G 93 ix

10 LIST F SCEMES SCEME PAGE Scheme 1 Pathway to (b 3 ) + (1a) and (b cat) + (1b) from AAAG through 5- membered cyclic intermediates...1 Scheme 2 Scheme 3 Scheme 4 Scheme 5 Scheme 6 Scheme 7 Scheme 8 Scheme 9 Multi-stage CID scheme of thallium cationized LGGFL..4 Multi-stage CID scheme of thallium cationized FGGLL..42 Multi-stage CID scheme of thallium cationized FGGGL..42 Multi-stage CID scheme of thallium cationized AGGFL..45 Multi-stage CID scheme of sodium cationized AGGFL...45 Multi-stage CID scheme of protonated AGGFL...46 Multi-stage CID scheme of silver cationized AGGFL..47 Reaction mechanism for formation of (b 3 ) + from A(4AMBz)AG...68 Scheme 1 Pathway to (b 3-1+cat) + from metal cationized AA(α-d 2 -γabu)g through 7- membered cyclic intermediate, with transfer of amide position atom...94 Scheme 11 Scheme 12 Potential pathway to (b 3-1+cat) + from metal cationized AA(α-d 2 -γabu)g through 7-membered cyclic intermediate, with transfer of α-carbon position atom.95 Pathway to (b 3-1+cat) + from metal cationized AA(α-d 2 -γabu)g through alternative ketene mechanism 96 x

11 LIST F TABLES TABLE Table 1 Table 2 PAGE Representative product ion distribution of protonated and metal cationized YGGFL, VGVAPG, WWLQL peptides.38 Representative product ion distributions of protonated and metal cationized GPA, FGG and GG peptides.39 xi

12 CAPTER 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 a n, and b n ions which are the -terminal fragments, and y n ions that are C-terminal fragments [1, 2] (figure 1.1). The reaction pathways leading to b n and y n 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- 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

13 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 -terminal side, thus forming a fivemembered ring intermediate. C- 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 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+) + ions. The major difference between the CID of alkali metal-cationized peptides and protonated peptides is that the former predominantly form (b n +17+cat) + while the latter forms b n and y n ions (figure 1.2). 2

14 a 1 b 1 a 2 b 2 b 3 a 3 R R 2 C C C C C C C C R R x 3 x 2 x 1 y 3 y2 y 1 Figure 1.1 Biemann nomenclature for fragment ions where b n type ions contain -terminus and y n type ions contain the C-terminus. R R 1 R R 4 R 5 b n + y n + Figure 1.2 representative structures of protonated b and y ions. The mechanism for formation of (b n +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 -terminal fragment ion, which can undergo the same fragmentation reaction multiple times in a multiple-stage CID experiment. Formation of (b n -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 -terminal side of the cleavage site( as shown for b + 3 from AAAG in 3

15 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 (b n +17+cat) + ions. At higher CID stages however, dissociation of the YG (b cat) + results in the production of (a 2-1+cat) + species. In the investigation of YGGFL it was found that (b 4-1+Ag) + was favored over both the (b Ag) + and (a 4-1+Ag) + ions at activation amplitude ranging from 2-3% of the excitation voltages accessible on the LCQ-Deca TM and an activation time of 3ms. The multiple-stage CID of Ag + cationized leucine enkephalin can be initiated with either the (b n -1+Ag) + or (b n +17+Ag) + produced at the MS/MS stage. Multiple stage CID of (b n -1+Ag) + proceeds through sequential elimination of C to produce (a n -1+Ag) + ions and then subsequent loss of imine of the C-terminal amino acid to generate the next (b 2-1+Ag) + species. Similar to the alkali cationized peptides, CID of (b 2-1+Ag) + produces prominent (a 2-1+Ag) + ions. It has been noted that (b 1 ) ions are not normally observed in the CID of protonated peptides and even with metal cationized peptides, formation of (b 1-1+cat) + is not the favored process. The lack of (b 1-1+cat) + is attributed to the fact that the mechanism for the generation of (b n ) products involves the intervention of a five membered ring structure. This particular mechanism requires cleavage of a carbonyl moiety to the -terminal side of the amide bond in the dissociation reaction. In the case of the (b 2-1+cat) + or (b cat) + species, such a carbonyl group is not present and CID of these precursor ions leads primarily to the production of the cationized imines (a 2-1+cat) +. 4

16 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 (b n -1+cat) + and (a n -1+cat) + ions which are similar to the CID of protonated peptides, while the production of (b n +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 (a n -1+cat) +, (b n -1+cat) + and (b n +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 a + ) cationized peptides. The former primarily form (b n -1+cat) + and the later forms (b n +17+cat) + as the major product ions respectively. For identical peptides, the product ions of (M+Ag) + are more intense than those of (M+a) +, and relative intensities of the (b n ++Ag) + ions are typically lower than those of the (b n ++a) + 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

17 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 (b n +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 2 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 (b n-1 +a +) + 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 (b n-1 +a+) + ion as the most abundant ion following CID, they do provide other alkali cationized analogs, such as b n-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

18 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 -acetylated tetrapeptides, with the general sequence AcFGGX, that featured C-termini designed to allow transfer of the required to generate the (b 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 a + - cationized AcFGGX, formation of (b 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), ε- caproic 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 b + n and y + n ions. This effect was attributed to the 7

19 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 b + n and y + n ions because the aromatic ring precluded cyclization and intramolecular nucleophililc attack, it also enhanced the formation of other b + + n ions. For example, increased intensities of b 3 and b + 2 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 (b 3-1+cat) + and (b 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 +, a + 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 (b 3-1+cat) + ion, primarily through 8

20 generation of the stable, highly conjugated oxazolinone, at the expense of the normally favored (b 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 (b 3-1+cat) + products from Li +, a + and Ag + cationized AAXG was investigated. The initial hypothesis was that the potential prohibitively large cyclic intermediates would suppress formation of (b 3-1+cat) +, as was observed for the protonated versions of the peptide. owever, the metal cations may coordinate with the peptide through interactions with multiple amide carbonyl atoms, and thus kinetically assist formation of a reactive configuration from which nulceophilic attack occurs. As we show here, (b 3-1+cat) + is a prominent, if not dominant, reaction product from the metal cationized AAXG. 9

21 Scheme 1a Scheme 1b 2 + Proton transfer 2 cat cat b C, =C 2 2 cat + cat + cat + (b cat) + Scheme 1 Pathway to (b 3 ) + (1a) and (b cat) + (1b) from AAAG through 5- membered cyclic intermediates. 1

22 1 8 (y 2 ) AA(βA)G R. I. (%) R. I. (%) (y 2 ) (y 2 ) (y 3 ) (b 3 ) (y 3 ) AA(γAbu)G AA(Cap)G R. I. (%) (y 3 ) R. I. (%) (y 2 ) (b 2 ) (b 3 ) (y 3 ) AA(4AMBz)G m/z (used with permission from reference 39). Figure 1.3 CID spectra of protonated AAXG. 11

23 CAPTER 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 197 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 [4]. 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

24 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

25 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 193 s carried out experiments using electrospray ionization. Some 3 years later (196), the pioneering work of Malcolm Dole et al demonstrated the use of electrospray to ionize intact chemical species. A further 2 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 22 obel Prize for chemistry. Fenn and co-workers in 198 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

26 small droplets about 1µm 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 2 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+] + (M= analyte molecule, = hydrogen ion). In other cases a metal cation such as sodium ion is used [M+a] + or the molecule can be deprotonated [M ] -. Also in electrospray, multiply charged ion such as [M+2] 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

27 Spray needle tip Solvent evaporation Rayleigh limit at this point +++ Multiply charged droplet Coulombic explosion. + ve Power supply -ve Analyte ions Counter electrodes Figure 2.2 Principle of electrospray ionization. 16

28 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 obel Prize winning mass spectrometry pioneer, Wolfgang Paul. is work in the early 195 s led to the development of the basic parameters of today s benchtop instruments [44]. owever it took breakthroughs in design at the Finnigan MAT in the 198 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 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

29 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 q z q z = 4eV/mr 2 ω 2 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 a z and q z. Depending upon the amplitude of the voltage placed on the ring electrode, an ion of a given m/z will have a z and q z values that are within the boundaries of the stability diagram and the ion will be trapped. If a z and q z values at that voltage fall outside the boundaries of the stability region, the ion will hit the electrode and will be lost. 18

30 Fundamental rf. Ring electrode ~ Endcap electrodes v Auxiliary ac. Figure 2.3 Basic components of quadrupole ion trap analyzer. 19

31 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 1-1eV range of collision energy and high energy collision seen in sector and TF/TF 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 2

32 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. owever, excitation in an ion trap is still fairly fast, due to the high pressure of the helium present in the trap ( 1-3 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+] +, [M-] - 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 MS 3 spectrum (figure 2.5). This process can be repeated a number of times, resulting in a series of MS n 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

33 Ion source Mass analyzer MS I Detector m/z Ion source Mass analyzer Collision cell Detector I I MS m/z MS/MS m/z Figure 2.4 Collision induced dissociation of precursor ions. 22

34 [M+X] + intensity Intensity Product ions Isolate and fragment Isolate and fragment m/z Figure 2.5 Principle of Tandem mass spectrometry 23

35 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 Li 3, a 3, Ag 3, Tl 3 and Glycine (G), alanine (A), and valine (V)-loaded Wang resins, Fmoc-alanine (Ala), β-alanine [ 2 C 2 C 2 C 2, βa], γ-aminobutyric acid [ 2 C 2 C 2 C 2 C 2, γabu], γ-aminobutyric acid labeled with deuterium at the α-carbon position [ 2 C 2 C 2 CD 2 C 2, α-d 2 -γabu], ε-aminocaproic acid [ 2 C 2 C 2 C 2 C 2 C 2 C 2, Cap] and 4-aminomethylbenzoic acid [ 2 C 2 C 6 4 C 2, 4AMBz] were purchased from Sigma-Aldrich (St. Louis, M) 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 PLC grade Me and de-ionized 2. Equimolar metal nitrate solutions were prepared in deionized 2 to produce the required final concentration. To investigate the dissociation of AAXG peptides for which exchangeable atoms were replaced with D, the peptide was incubated in a mixture (5:5 by volume) of D 2 and C 3 D (Aldrich Chemical, St. Louis M) 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 LCQ- Deca TM ion trap mass spectrometer (Thermoquest Corporation, San Jose, CA, USA) 24

36 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 2 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 22 c. This served as desolvation temperature to convert ions from solution phase to gas phase. The ion-trap analyzer was operated at 1x 1-6 Torr. elium 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 1 to 3% 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.3. The activation time employed at each CID stage was set at 3ms. 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/3) x (.4 + (.2 x Precursor ion mass ) CAA= (AA/3) x (.4 + (.2 x Precursor ion mass )/(3-6). 25

37 Where CAA is the corrected activation amplitude, AA is the normalized collision energy and 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 (b 3-1+cat) + and (b cat) + for peptides with and without the 4AMBz residue. For ion isolation experiments to probe D for back exchange, precursor ions were isolated using an isolation width of m/z units, which was sufficient to isolate a single isotopic peak. The normalized collision energy was set at % (i.e. no imposed collisional activation), the activation Q at.3, and the isolation time altered from 1 msec to 1 second. During the isolation period the ions may collide with 2, which is present as a contaminant in the ion trap and because of its use as solvent for ESI. The check for D for back exchange was done because such a process could effect the splitting of (b 3-1+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 or D atoms during fragmentation reactions. 26

38 Figure 2.6 Multiple reaction vessel peptide synthesis apparatus. 27

39 CAPTER 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. owever, 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 -terminus containing (b-type) rearrangement ion labeled (b n +17+cat) + or (b n ++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 +, a + ) cationized peptides [27], including the fact that the most prominent species observed during the CID of alkali metal(li +, a + ) cationized leucine enkephalin 28

40 are the (b n +17+cat) + ions, while Ag + cationized peptides favor formation of (b n -1+Ag) + over both the (b n +17+Ag) + and (a n -1+Ag) + ions. Similar to the alkali cationized peptides, multi-stage CID of (b 2-1+Ag) + produces prominent (a 2-1+Ag) + ions. While the differences between Ag + and alkali-metal (Li +, a + ) 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 +, a +, 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 b 3 (242) and y + 2 (237) product ions. The difference in m/z values between the (M+) + and b + 3 is 165u (47-242) thus identifying + phenylalanine (147u) and 2 (18u). The m/z value resulting in the formation of y 2 (237) was 17u which is consistent with the loss of -terminus residues valine (99u) and alanine (71u). For a + cationized VAAF, the peak with the largest m/z value was (b a) + (282u). The amino acid cleaved from the C-terminus was identified as phenylalanine (147u), since (M+a) + had an m/z value of 429u. The Ag + cationized 29

41 version generated (b 3-1+Ag) + (348,35) as the major product ion more than (a Ag) + (32,322) and (b Ag) + (366,368) species. Silver ion has two stable isotopes 17 Ag and 19 Ag 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 (b Tl) + having a mass unit of 462u and 464u due to two isotopes of thallium ( 25 Tl and 27 Tl). ther product ions observed were, (b 3-1+Tl) + (444, 446) and (b 2-1+Tl) + (373, 375). ne striking observation is that, among the metal cationized peptide (VAAF) investigated Tl + cationized peptide generated greater abundance of (b 2-1+Cat) + ions. The most prominent product ion generated from the CID of protonated YGGFL figure 3.2, was b + 4 (425u) ion due to the neutral loss of leucine (113). The other product ion observed was a + 4 (397u). In contrast to CID of protonated peptide, CID of a + cationized YGGFL generated (b a) + in abundance. ther minor products obtained were (b 4-1+a) + and (a 4-1+a) + ions. The remarkable differences between the protonated peptides and a + 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 (b 4-1+Ag) +, (a 4-1+Ag) + and (b Ag) + were observed in the CID spectra of Ag + cationized YGGFL. It is interesting that the yield of (b Ag) + ion from YGGFL was approximately 35%(figure 3.2c), significantly higher than the corresponding ions 3

42 (b 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 a + cationized versions, the most prominent product ion resulting from the CID of Tl + cationized leucine enkephalin was (b 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 (b 4-1+Tl) + had the highest peak intensity. This trend in which the major product ion was either (b n +17+Tl) + or (b n -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) a +, (c) Ag + and (d) Tl + cationized forms. For this peptide, each cation demonstrated a unique pattern of fragmentation. ne interesting observation was that there was high abundance of (a 4-1+Tl) + compared to (a 4-1+Ag) + among the two metal cationized peptides. The spectra shown in figures 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 +, a + ) versions of the peptides did not generate prominent (b n -1+cat) + ions. The different behaviors of Ag + and a + metal ions are strikingly consistent with their differences in hardness as lewis acids, a + prefers to interact with the harder (oxygen) donors, whereas Ag + prefers to bind to softer (nitrogen) donors. It is interesting then, that generation of (b n -1+cat) + which involves transfer of proton ultimately to an - atom is the preferred pathway for Ag + cationized peptides. The loss of 2, which involves 31