N-terminal derivatization and fragmentation of neutral peptides via ion--molecule reactions with acylium ions: toward gas-phase Edman degradation?

The gas-phase ion-molecule reactions of neutral alanylglycine have been examined with various mass-selected acylium ions RCO(+) (R= CH(3), CD(3), C(6)H(5), C(6)F(5) and (CH(3))( 2)N), as well as the transacylation reagent O-benzoylbenzophenone in a Fourier transform ion cyclotron resonance mass spectrometer. Reactions of the gaseous dipeptide with acylium ions trapped in the ICR cell result in the formation of energized [M + RCO](+) adduct ions that fragment to yield N-terminal b-type and C-terminal y-type product ions, including a modified b(1) ion which is typically not observed in the fragmentation of protonated peptides. Judicious choice of the acylium ion employed allows some control over the product ion types that are observed (i.e., b versus y ions). The product ion distributions from these ion--molecule reactions are similar to those obtained by collision-activated dissociation in a triple quadrupole mass spectrometer of the authentic N-acylated alanylglycine derivatives. These data indicate that derivatization of the peptide in the gas-phase occurs at the N-terminal amine. Ab initio molecular orbital calculations, performed to estimate the thermochemistry of the steps associated with adduct formation as well as product ion formation, indicate that (i) the initially formed adduct is energized and hence likely to rapidly undergo fragmentation, and (ii) the likelihood for the formation of modified b(1) ions in preference to y(1) ions is dependent on the R substituent of the acylium ion. The reaction of the tetrapeptide valine--alanine--alanine--phenylalanine with the benzoyl cation was also found to yield a number of product ions, including a modified b(1) ion. This result suggests that the new experimental approach described here may provide a tool to address one of the major limitations associated with traditional mass spectrometric peptide sequencing approaches, that is, determination of the identity and order of the two N-terminal amino acids. Analogies are made between the reactions observed here and the derivatization and N-terminal cleavage reactions employed in the condensed-phase Edman degradation method.     

Investigation of c ions formed by N‐terminally charged peptides upon collision‐induced dissociation

Peptide fragments such as b and y sequence ions generated upon low‐energy collision‐induced dissociation have been routinely used for tandem mass spectrometry (MS/MS)‐based peptide/protein identification. The underlying formation mechanisms have been studied extensively and described within the literature. As a result, the ‘mobile proton model’ and ‘pathways in competition model’ have been built to interpret a majority of peptide fragmentation behavior. However, unusual peptide fragments which involve unfamiliar fragmentation pathways or various rearrangement reactions occasionally appear in MS/MS spectra, resulting in confused MS/MS interpretations. In this work, a series of unfamiliar c ions are detected in MS/MS spectra of the model peptides having an N‐terminal Arg or deuterohemin group upon low‐energy collision‐induced dissociation process. Both the protonated Arg and deuterohemin group play an important role in retention of a positive charge at the N‐terminus that is remote from the cleavage sites. According to previous reports and our studies involving amino acid substitutions and hydrogen–deuterium exchange, we propose a McLafferty‐type rearrangement via charge‐remote fragmentation as the potential mechanism to explain the formation of c ions from precursor peptide ions or unconventional b ions. Density functional theory calculations are also employed in order to elucidate the proposed fragmentation mechanisms. Copyright © 2016 John Wiley & Sons, Ltd.     

Comparison of CID,ETD and metastable atom‐activated dissociation (MAD) of doubly and triply charged phosphorylated tau peptides

The fragmentation behavior of the 2+ and 3+ charge states of eleven different phosphorylated tau peptides was studied using collision‐induced dissociation (CID), electron transfer dissociation (ETD) and metastable atom‐activated dissociation (MAD). The synthetic peptides studied contain up to two known phosphorylation sites on serine or threonine residues, at least two basic residues, and between four and eight potential sites of phosphorylation. CID produced mainly b‐/y‐type ions with abundant neutral losses of the phosphorylation modification. ETD produced c‐/z‐type ions in highest abundance but also showed numerous y‐type ions at a frequency about 50% that of the z‐type ions. The major peaks observed in the ETD spectra correspond to the charge‐reduced product ions and small neutral losses from the charge‐reduced peaks. ETD of the 2+ charge state of each peptide generally produced fewer backbone cleavages than the 3+ charge state, consistent with previous reports. Regardless of charge state, MAD achieved more extensive backbone cleavage than CID or ETD, while retaining the modification(s) in most cases. In all but one case, unambiguous modification site determination was achieved with MAD. MAD produced 15–20% better sequence coverage than CID and ETD for both the 2+ and 3+ charge states and very different fragmentation products indicating that the mechanism of fragmentation in MAD is unique and complementary to CID and ETD. Copyright © 2012 John Wiley & Sons, Ltd.     

Observation of an unusually facile fragmentation pathway of gas-phase peptide ions: a study on the gas-phase fragmentation mechanism and energetics of tryptic peptides modified with 4-sulfophenyl isothiocyanate (SPITC) and 4-chlorosulfophenyl isocyanate (SPC) and their 18-crown-6 complexes

Various peptide modifications have been explored recently to facilitate the acquisition of sequence information. N-terminal sulfonation is an interesting modification because it allows unambiguous de novo sequencing of peptides, especially in conjunction with MALDI-PSD-TOF analysis; such modified peptide ions undergo fragmentation at energies lower than those required conventionally for unmodified peptide ions. In this study, we systematically investigated the fragmentation mechanisms of N-terminal sulfonated peptide ions prepared using two different N-terminal sulfonation reagents: 4-sulfophenyl isothiocyanate (SPITC) and 4-chlorosulfophenyl isocyanate (SPC). Collision-induced dissociation (CID) of the SPC-modified peptide ions produced a set of y-series ions that were more evenly distributed relative to those observed for the SPITC-modified peptides; y(n-1) ion peaks were consistently and significantly larger than the signals of the other y-ions. We experimentally investigated the differences between the dissociation energies of the SPITC- and SPC-modified peptide ions by comparing the MS/MS spectra of the complexes formed between the crown ether 18-crown-6 (CE) and the modified peptides. Upon CID, the complexes formed between 18-crown-6 ether and the protonated amino groups of C-terminal lysine residues underwent either peptide backbone fragmentation or complex dissociation. Although the crown ether complexes of the unmodified ([M + CE + 2H]2+) and SPC-modified ([M* + CE + 2H]2+) peptides underwent predominantly noncovalent complex dissociation upon CID, the low-energy dissociations of the crown ether complexes of the SPITC-modified peptides ([M' + CE + 2H]2+) unexpectedly resulted in peptide backbone fragmentations, along with a degree of complex dissociation. We performed quantum mechanical calculations to address the energetics of fragmentations observed for the modified peptides.     

Formation of [b(n−1)+OH+H]+ ion structural analogs by solution-phase chemistry

Derivatization of a variety of peptides by a method known to enhance anhydride formation is demonstrated by mass spectrometry to yield ions that have elemental composition and fragmentation properties identical to [b(n-1) + OH + H]+ ions formed by gas-phase rearrangement and fragmentation. The [b(n-1) + OH + H]+ ions formed by gas-phase rearrangement and fragmentation and the solution-phase [b(n-1) + OH + H]+ ion structural analogs formed by derivatization chemistry show two different forms of dissociation using multiple-collision CAD in a quadrupole ion trap and unimolecular decomposition in a TOF-TOF; one group yields identical product ions as a truncated form of the peptide with a free C-terminal carboxylic acid and fragments at the same activation energy; the other group fragments differently from the truncated peptide, being more resistant to fragmentation than the truncated peptide and yielding primarily the [b(n-2) + OH + H]+ product ion. Nonergodic electron capture dissociation MS/MS suggests that any structural differences between the specific-fragmenting [b(n-1) + OH + H]+ ions and the truncated peptide is at the C-terminus of the peptide. The specific-fragmentation can be readily observed by MS(n) experiments to occur in an iterative fashion, suggesting that the C-terminal structure of the original [b(n-1) + OH + H]+ ion is maintained after subsequent rearrangement and fragmentation events in peptides which fragment specifically. A mechanism for the formation of specific-fragmenting and nonspecific-fragmenting [b(n-1) + OH + H]+ ions is proposed.     

Mechanisms for the selective gas-phase fragmentation reactions of methionine side chain fixed charge sulfonium ion containing peptides

To enable the development of improved tandem mass spectrometry based methods for selective proteome analysis, the mechanisms, product ion structures, and other factors influencing the gas-phase fragmentation reactions of methionine side-chain derivatized "fixed-charge" phenacylsulfonium ion containing peptide ions have been examined. Dissociation of these peptide ions results in the exclusive characteristic loss of the derivatized side chain, thereby enabling their selective identification. The resultant product ion(s) are then subjected to further dissociation to obtain sequence information for subsequent protein identification. Molecular orbital calculations (at the B3LYP/6-31 + G (d,p) level of theory) performed on a simple peptide model, together with experimental evidence obtained by multistage dissociation of a regioselectively deuterated methionine derivatized sulfonium ion containing tryptic peptide, indicate that fragmentation of the fixed charge containing peptide ions occurs via SN2 reactions involving the N- and C-terminal amide bonds adjacent to the methionine side chain, resulting in the formation of stable cyclic five- and six-membered iminohydrofuran and oxazine product ions, respectively. These studies further indicate that the rings formed via these neighboring group reactions are stable to further dissociation by MS3. As a consequence, the formation of b- or y-type sequence ions are "skipped" at the site of cyclization. Despite this, complete sequence information is still obtained because of the presence of both cyclic products.     

Identification and localization of the fatty acid modification in ghrelin by electron capture dissociation

Electron capture dissociation (ECD) has been demonstrated to be an effective fragmentation technique for characterizing the site and structure of the fatty acid modification in ghrelin, a 28-residue growth-hormone-releasing peptide that has an unusual ester-linked n-octanoyl (C8:0) modification at Ser-3. ECD cleaves 21 of 23 possible backbone amine bonds, with the product ions (c and z· ions) covering a greater amino acid sequence than those obtained by collisionally activated dissociation (CAD). Consistent with the ECD nonergodic mechanism, the ester-linked octanoyl group is retained on all backbone cleavage product ions, allowing for direct localization of this labile modification. In addition, ECD also induces the ester bond cleavage to cause the loss of octanoic acid from the ghrelin molecular ion; the elimination process is initiated by the capture of an electron at the protonated ester group, which is followed by the radical-site-initiated reaction known as -cleavage. The chemical composition of the attached fatty acid can be directly obtained from the accurate Fourier transform ion cyclotron resonance (FTICR) mass measurement of the ester bond cleavage product ions.     

Metastable atom‐activated dissociation mass spectrometry: leucine/isoleucine differentiation and ring cleavage of proline residues

Extensive backbone fragmentation resulting in a‐, b‐, c‐, x‐, y‐ and z‐type ions is observed of singly and doubly charged peptide ions through their interaction with a high kinetic energy beam of argon or helium metastable atoms in a modified quadrupole ion trap mass spectrometer. The ability to determine phosphorylation‐sites confirms the observation with previous reports and we report the new ability to distinguish between leucine and isoleucine residues and the ability to cleave two covalent bonds of the proline ring resulting in a‐, b‐, x‐, y‐, z‐ and w‐type ions. The fragmentation spectra indicate that fragmentation occurs through nonergodic radical ion chemistry akin to electron capture dissociation (ECD), electron transfer dissociation (ETD) and electron ionization dissociation mechanisms. However, metastable atom‐activated dissociation mass spectrometry demonstrates three apparent benefits to ECD and ETD: (1) the ability to fragment singly charged precursor ions, (2) the ability to fragment negatively charged ions and (3) the ability to cleave the proline ring that requires the cleavage of two covalent bonds. Helium metastable atoms generated more fragment ions than argon metastable atoms for both substance P and bradykinin regardless of the precursor ion charge state. Reaction times less than 250 ms and efficiencies approaching 5% are compatible with on‐line fragmentation, as would be desirable for bottom‐up proteomics applications. Copyright © 2009 John Wiley & Sons, Ltd.     

Fragmentation of peptide disulfides under conditions of negative ion mass spectrometry: Studies of oxidized glutathione and contryphan

The fragmentation of positive and negative ions of peptide disulfides under mass spectrometric conditions yields distinctly different product ion distributions. A negative ion upon collision induced dissociation yields intense product ions, which correspond to cleavage at the disulfide linkage. The complete assignment of the product ions obtained upon fragmentation of oxidized glutathione in an ion trap is presented. The cleavage at the disulfide site is mediated by abstraction of CalphaH and CbetaH protons resulting in product ions derived by neutral loss of H2S2 and H2S. The formation of peptide thioaldehydes and persulfides at the cysteine sites is established. Dehydroalanine formation at the Cys residue is predominant. The case of a contryphan, a cyclic peptide disulfide derived from Conus snail venom, illustrates the utility of negative ion mass spectrometry in disulfide identification. Complementary information is derived by combining the fragmentation patterns obtained from positive and negative ions of disulfide containing peptides.     

Infrared multiphoton dissociation spectroscopic analysis of peptides and oligosaccharides by using fourier transform ion cyclotron resonance mass spectrometry with a midinfrared free-electron laser

The fragmentation of peptides and oligosaccharides in the gas phase was investigated by means of electrospray ionization Fourier transform ion cyclotron resonance (FTICR) mass spectrometry coupled with dissociation by a laser-cleavage infrared multiphoton dissociation (IRMPD) technique. In this technique, an IR free-electron laser is used as a tunable source of IR radiation to cause cleavage of the ionized samples introduced into the FTICR cell. The gas-phase IRMPD spectra of protonated peptides (substance P and angiotensin II) and two sodiated oligosaccharides (sialyl Lewis X and lacto-N-fucopentaose III) were obtained over the IR scan range of 5.7-9.5 microm. In the IRMPD spectra for the peptide, fragment ions are observed as y/b-type fragment ions in the range 5.7-7.5 microm, corresponding to cleavage of the backbone of the parent amino acid sequence, whereas the spectra of the oligosaccharides have major peaks in the range 8.4-9.5 microm, corresponding to photoproducts of the B/Y type.     

Formation of c1 fragment ions in collision-induced dissociation of glutamine-containing peptide ions: a tip for de novo sequencing

A c1 ion was observed with significant yield in the tandem mass (MS/MS) spectra of peptide ions containing glutamine as the second amino acid residue from the N-terminus. The c1 fragment was generated independently of the N-terminal residue of the peptide, but its abundance was strongly dependent on the side-chain identity. This ion is not a common fragmentation product in low-energy collision-induced dissociation of peptide ions, but it assists in identification of the first two amino acid residues, often difficult due to a low or absent signal from the heaviest y ion. A consecutive fragmentation mechanism is proposed, involving a b2 ion with a six-membered ring as an intermediate, to explain the exceptional stability of the c1 fragment ion. The utility of this information is discussed, especially in de novo sequencing of peptide ions.     

Substituent effects on the gas-phase fragmentation reactions of sulfonium ion containing peptides

The multistage mass spectrometric (MS/MS and MS3) gas-phase fragmentation reactions of methionine side-chain sulfonium ion containing peptides formed by reaction with a series of para-substituted phenacyl bromide (XBr where X=CH2COC6H4R, and R=--COOH, --COOCH3, --H, --CH3 and --CH2CH3) alkylating reagents have been examined in a linear quadrupole ion trap mass spectrometer. MS/MS of the singly (M+) and multiply ([M++nH](n+1)+) charged precursor ions results in exclusive dissociation at the fixed charge containing side chain, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility). However, loss of the methylphenacyl sulfide side-chain fragment as a neutral versus charged (protonated) species was observed to be highly dependent on the proton mobility of the precursor ion, and the identity of the phenacyl group para-substituent. Molecular orbital calculations were performed at the B3LYP/6-31+G** level of theory to calculate the theoretical proton affinities of the neutral side-chain fragments. The log of the ratio of neutral versus protonated side-chain fragment losses from the derivatized side chain were found to exhibit a linear dependence on the proton affinity of the side-chain fragmentation product, as well as the proton affinities of the peptide product ions. Finally, MS3 dissociation of the nominally identical neutral and protonated loss product ions formed by MS/MS of the [M++H]2+ and [M++2H]3+ precursor ions, respectively, from the peptide GAILM(X)GAILK revealed significant differences in the abundances of the resultant product ions. These results suggest that the protonated peptide product ions formed by gas-phase fragmentation of sulfonium ion containing precursors in an ion trap mass spectrometer do not necessarily undergo intramolecular proton 'scrambling' prior to their further dissociation, in contrast to that previously demonstrated for peptide ions introduced by external ionization sources.     

Pseudo-MS3 in a MALDI orthogonal quadrupole-time of flight mass spectrometer

Both the matrix selected and the laser fluence play important roles in MALDI-quadrupole/time of flight (QqTOF) fragmentation processes. "Hot" matrices, such as alpha-cyano4-hydroxycinnamic acid (HCCA), can increase fragmentation in MS spectra. Higher laser fluence also increases fragmentation. Typical peptide fragment ions observed in the QqTOF are a, b, and y ion series, which resemble low-energy CID product ions. This fragmentation may occur in the high-pressure region before the first mass-analyzing quadrupole. Fragment ions can be selected by the first quadrupole (Q1), and further sequenced by conventional MS/MS. This allows pseudo-MS3 experiments to be performed. For peptides of higher molecular weight, pseudo-MS3 can extend the mass range beyond what is usually accessible for sequencing, by allowing one to sequence a fragment ion of lower molecular weight instead of the full-length peptide. Peptides that predominantly show a single product ion after MS/MS yield improved sequence information when this technique is applied. This method was applied to the analysis of an in vitro phosphorylated peptide, where the intact enzymatically-generated peptide showed poor dissociation via MS/MS. Sequencing a fragment ion from the phosphopeptide enabled the phosphorylation site to be unambiguously determined.     

Phosphorylated serine and threonine residues promote site‐specific fragmentation of singly charged,arginine‐containing peptide ions

In order to investigate gas‐phase fragmentation reactions of phosphorylated peptide ions, matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass (MS/MS) spectra were recorded from synthetic phosphopeptides and from phosphopeptides isolated from natural sources. MALDI‐TOF/TOF (TOF: time‐of‐flight) spectra of synthetic arginine‐containing phosphopeptides revealed a significant increase of y ions resulting from bond cleavages on the C‐terminal side of phosphothreonine or phosphoserine. The same effect was found in ESI‐MS/MS spectra recorded from the singly charged but not from the doubly charged ions of these phosphopeptides. ESI‐MS/MS spectra of doubly charged phosphopeptides containing two arginine residues support the following general fragmentation rule: Increased amide bond cleavage on the C‐terminal side of phosphorylated serines or threonines mainly occurs in peptide ions which do not contain mobile protons. In MALDI‐TOF/TOF spectra of phosphopeptides displaying N‐terminal fragment ions, abundant b–H₃PO₄ ions resulting from the enhanced dissociation of the pSer/pThr–X bond were detected (X denotes amino acids). Cleavages at phosphoamino acids were found to be particularly predominant in spectra of phosphopeptides containing pSer/pThr–Pro bonds. A quantitative evaluation of a larger set of MALDI‐TOF/TOF spectra recorded from phosphopeptides indicated that phosphoserine residues in arginine‐containing peptides increase the signal intensities of the respective y ions by almost a factor of 3. A less pronounced cleavage‐enhancing effect was observed in some lysine‐containing phosphopeptides without arginine. The proposed peptide fragmentation pathways involve a nucleophilic attack by phosphate oxygen on the carbon center of the peptide backbone amide, which eventually leads to cleavage of the amide bond. Copyright © 2009 John Wiley & Sons, Ltd.     

Gas-phase fragmentation characteristics of benzyl-aminated lysyl-containing tryptic peptides

The fragmentation characteristics of peptides derivatized at the side-chain ε-amino group of lysyl residues via reductive amination with benzaldehyde have been examined using collision-induced dissociation (CID) tandem mass spectrometry. The resulting MS/MS spectra exhibit peaks representing product ions formed from two independent fragmentation pathways. One pathway results in backbone fragmentation and commonly observed sequence ion peaks. The other pathway corresponds to the unsymmetrical, heterolytic cleavage of the C_ζ-N_ε bond that links the benzyl derivative to the side-chain lysyl residue. This results in the elimination of the derivative as a benzylic or tropylium carbocation and a (n − l)⁺-charged peptide product (where n is the precursor ion charge state). The frequency of occurrence of the elimination pathway increases with increasing charge of the precursor ion. For the benzylmodified tryptic peptides analyzed in this study, peaks representing products from both of these pathways are observed in the MS/MS spectra of doubly-charged precursor ions, but the carbocation elimination pathway occurs almost exclusively for triply-charged precursor ions. The experimental evidence presented herein, combined with molecular orbital calculations, suggests that the elimination pathway is a charge-directed reaction contingent upon protonation of the secondary ε-amino group of the benzyl-derivatized lysyl side chain. If the secondary ε-amine is protonated, the elimination of the carbocation is observed. If the precursor is not protonated at the secondary ε-amine, backbone fragmentation persists. The application of appropriately substituted benzyl analogs may allow for selective control over the relative abundance of product ions generated from the two pathways.     

Dissociation pathways of alkali-cationized peptides: Opportunities for C-terminal peptide sequencing

Dissociation pathways of alkali-cationized peptides have been studied using multiple stages of mass spectrometry (MSx) with a quadrupole ion trap mass spectrometer. Over 100 peptide ions ranging from 2 to 10 residues in length and containing each of the 20 common amino acids have been examined. The formation of the [b(n-1) + Na + OH]+ product ion is the predominant dissociation pathway for the majority of the common amino acids. This product corresponds to a sodium-cationized peptide one residue shorter in length than the original peptide. In a few cases, product ions such as [b(n-1) + Na - H]+ and those formed by loss, or partial loss, of a sidechain are observed. Both [b(n-1) + Na + OH]+ and [b(n-1) + Na - H]+ product ions can be selected as parent ions for a subsequent stage of tandem mass spectrometry (MS/MS). It is shown that these dissociation patterns provide opportunities for peptide sequencing by successive dissociation from the C-terminus of alkali-cationized peptides. Up to seven stages of MS/MS have been performed on a series of [b + Na + OH]+ ions to provide sequence information from the C-terminus. This method is analogous to Edman degradation except that the cleavage occurs from the C-terminus instead of the N-terminus, making it more attractive for N-terminal blocked peptides. The isomers leucine and isoleucine cannot be differentiated by this method but the isobars lysine and glutamine can.     

Neutral fragment mass spectra via ambient thermal dissociation of peptide and protein ions

A novel method for the fragmentation of peptide and protein ions at atmospheric pressure outside the mass spectrometer is described. Peptide/protein ions generated by electrosonic spray ionization (ESSI) are carried through a heated coiled metal tube where they fragment. Fragment ions of types a, b, and y are observed for peptides such as angiotensin II and bradykinin. In the case of phosphopeptides, informative b and y ions which preserve the labile phosphate groups are observed in the negative ion mode, which is potentially useful in the location of phosphorylation sites in proteins through chemical analysis of phosphopeptides. The thermal dissociation method extends to proteins such as ubiquitin and myoglobin, giving rise to y-type and other fragment ions. The most important feature of this method is that it also allows characterization of the neutral fragments arising from thermal dissociation by use of on-line corona discharge ionization. This neutral re-ionization experiment is much easier to perform outside the mass spectrometer than as conventionally done, in vacuum. It yields increased structural information from the resulting mass spectra in both the positive and the negative ion modes.     

Fragmentation of oligoribonucleotides from gas-phase ion-electron reactions

We have recently demonstrated that both electron capture dissociation (ECD) and electron detachment dissociation (EDD) can provide complementary sequence-specific cleavage of DNA compared with collision activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD). However, EDD is preferred because of more extensive fragmentation at higher sensitivity (due to its negative ion mode operation). Here, we extend the radical ion chemistry of these two gas-phase ion-electron reaction techniques to the characterization of RNA. Compared with DNA, rather limited information is currently available on the gas-phase fragmentation of RNA. We found that the ECD fragmentation patterns of the oligoribonucleotides A6, C6, and CGGGGC are nucleobase dependent, suggesting that cleavage proceeds following electron capture at the nucleobases. Only limited backbone cleavage was observed in ECD. EDD, on the other hand, provided complete sequence coverage for the RNAs A6, C6, G6, U6, CGGGGC, and GCAUAC. The EDD fragmentation patterns were different from those observed with CAD and IRMPD in that the dominant product ions correspond to d- and w-type ions rather than c- and y-type ions. The minimum differences between oligoribonucleotides suggest that EDD proceeds following direct electron detachment from the phosphate backbone.     

Energetics and dynamics of the fragmentation reactions of protonated peptides containing methionine sulfoxide or aspartic acid via energy- and time-resolved surface induced dissociation

The surface-induced dissociation (SID) of six model peptides containing either methionine sulfoxide or aspartic acid (GAILM(O)GAILR, GAILM(O)GAILK, GAILM(O)GAILA, GAILDGAILR, GAILDGAILK, and GAILDGAILA) have been studied using a specially configured Fourier transform ion-cyclotron resonance mass spectrometer (FT-ICR MS). In particular, we have investigated the energetics and dynamics associated with (i) preferential cleavage of the methionine sulfoxide side chain via the loss of CH3SOH (64 Da), and (ii) preferential cleavage of the amide bond C-terminal to aspartic acid. The role of proton mobility in these selective bond cleavage reactions was examined by changing the C-terminal residue of the peptide from arginine (nonmobile proton conditions) to lysine (partially mobile proton conditions) to alanine (mobile proton conditions). Time- and energy-resolved fragmentation efficiency curves (TFECs) reveal that selective cleavages due to the methionine sulfoxide and aspartic acid residues are characterized by slow fragmentation kinetics. RRKM modeling of the experimental data suggests that the slow kinetics is associated with large negative entropy effects and these may be due to the presence of rearrangements prior to fragmentation. It was found that the Arrhenius pre-exponential factor (A) for peptide fragmentations occurring via selective bond cleavages are 1-2 orders of magnitude lower than nonselective peptide fragmentation reactions, while the dissociation threshold (E0) is relatively invariant. This means that selective bond cleavage is kinetically disfavored compared to nonselective amide bond cleavage. It was also found that the energetics and dynamics for the preferential loss of CH3SOH from peptide ions containing methionine sulfoxide are very similar to selective C-terminal amide bond cleavage at the aspartic acid residue. These results suggest that while preferential cleavage can compete with amide bond cleavage energetically, dynamically, these processes are much slower compared to amide bond cleavage, explaining why these selective bond cleavages are not observed if fragmentation is performed under mobile proton conditions. This study further affirms that fragmentation of peptide ions in the gas phase are predominantly governed by entropic effects.     

The relative charge ratio between C and N atoms in amide bond acts as a key factor to determine peptide fragment efficiency in different charge states

The influence of charge state on the peptide dissociation behavior in tandem mass spectrometry (MS/MS) is worthy of discussion. Comparative studies of singly- and doubly-protonated peptide molecules are performed to explore the effect and mechanism of charge state on peptide fragmentation. In view of the charge-directed cleavage of protonated peptides described in the mobile proton model, radiolytic oxidation was applied to change the charge distribution of peptides but retain the sequence. Experimental studies of collision energy-dependent fragmentation efficiencies coupled with quantum chemical calculations indicated that the cleavage of ARRA and its side-chain oxidation products with oxygen atoms added followed a trend that doubly-protonated peptides fragment more easily than singly-protonated forms, while the oxidation product with the guanidine group deleted showed the opposite trend. By analyzing the charge distribution around the amide bonds, we found that the relative charge ratios between C and N atoms (Q_C/Q_N) in the amide bonds provided a reasonable explanation for peptide fragmentation efficiencies. An increase of the Q_C/Q_N value of the amide bond means that a peptide fragments more easily, and vice versa. The results described in this paper provide an experimental and calculation strategy for predicting peptide fragmentation efficiency.