Tandem electrospray mass spectrometric studies of proton and sodium ion adducts of neutral peptides with modified N- and C-termini: synthetic model peptides and microheterogeneous peptaibol antibiotics

The fragmentations of [M+H]+ and [M+Na]+ adducts of neutral peptides with blocked N- and C-termini have been investigated using electrospray ion trap mass spectrometry. The N-termini of these synthetically designed peptides are blocked with a tertiarybutyloxycarbonyl (Boc) group, and the C-termini are esterified. These peptides do not possess side chains that are capable of complexation and hence the backbone amide units are the sole sites of protonation and metallation. The cleavage patterns of the protonated peptides are strikingly different from those of sodium ion adducts. While the loss of the N-terminal blocking group occurs quite readily in the case of MS/MS of [M+Na]+, the cleavage of the C-terminal methoxy group seems to be a facile process in the case of MS/MS of [M+H]+ * Fragmentation of the protonated adducts yields only bn ions, while yn and a(n) ions are predominantly formed from the fragmentation of sodium ion adducts. The a(n) ions arising from the fragmentation of [M+Na](+) lack the N-terminal Boc group (and are here termed a(n)* ions). MS/MS of [M+Na]+ species also yields b(n) ions of substantially lower intensities that lack the N-terminal Boc group (b(n)*). A similar distinction between the fragmentation patterns of proton and sodium ion adducts is observed in the case of peptides possessing an N-terminal acetyl group. An example of the fragmentation of the H+ and Na+ adducts of a naturally occurring peptaibol from a Trichoderma species confirms that fragmentation of these two ionized species yields complementary information, useful in sequencing natural peptides. Inspection of the isotopic pattern of b(n) ions derived from [M+H]+ adducts of peptaibols provided insights into the sequences of microheterogeneous samples. This study reveals that the combined use of protonated and sodium ion adducts should prove useful in de novo sequencing of peptides, particularly of naturally occurring neutral peptides with modified N- and C-termini, for example, peptaibols.     

Fragmentation reactions of deprotonated peptides containing proline. The proline effect

The collision-induced dissociation (CID) fragmentation reactions of a variety of deprotonated peptides containing proline have been studied in detail using MS(2) and MS(3) experiments, deuterium labelling and accurate mass measurements when necessary. The [M--H--CO(2)](-) (a(2)) ion derived from H-Pro-Xxx-OH dipeptides shows an unusual fragmentation involving loss of C(2)H(4); this fragmentation reaction is not observed for larger peptides. The primary fragmentation reactions of deprotonated tripeptides with an N-terminal proline are formation of a(3) and y(1) ions. When proline is in the central position of tripeptides, a(3), y(2) and y(1) ions are the primary fragmentation products of [M--H](-), while when the proline is in the C-terminal position, a(3)and y(1) ions are the major primary products. In the latter case, the a(3) ion fragments primarily to the 'b(2) ion; further evidence is presented that the 'b(2) ions have a deprotonated oxazolone structure. Larger deprotonated peptides having at least two amino acid residues N-terminal to proline show a distinct preference for cleavage of the amide bond N-terminal to proline to form, mainly, the appropriate y ion. This proline effect is compared and contrasted with the similar proline effect observed in the fragmentation of protonated peptides containing proline.     

Electrospray ionization tandem mass spectrometric study on the effect of N-terminal beta- and gamma-carbo amino acids on fragmentation of GABA-hybrid peptides

The fragmentations of protonated and deprotonated ions of a new class of N-blocked hybrid Boc-carbopeptides containing repeats of gamma-Caa/gammaAbu- and beta-Caa/gammaAbu- (Caa==C-linked carbo gamma(4)-/beta(3)- amino acids derived from D-xylose, gammaAbu = gamma-aminobutyric acid) have been studied using electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). MS/MS of a pair of these protonated diastereomers produces distinct fragmentation of the Boc group. The formation of [M + H-56](+) corresponding to loss of isobutylene is more pronounced for Boc-NH-(R)-gamma-Caa-gammaAbu-OH (2) whereas it is of low abundance for Boc-NH-(S)-gamma-Caa-gammaAbu--OH (1). Similarly, MS(2) of [M--H](-) of 2 produces an abundant [M--H--C(CH(3))(3)OH--CO(2)](-) ion, which is absent for its diastereomeric isomer 1. From this, it can be suggested that MS/MS of N-blocked Boc-protected carbopeptides may be helpful in distinguishing the stereochemistry of the N-terminus Caa. MS(3) of [M + H-Boc + H](+) ions of peptides with a gamma-amino acid (gamma-Caa/gammaAbu) at the N-terminus produces only abundant y(n) (+) ions. On the other hand, characteristic fragmentations involving the peptide backbone (b(n) (+) and y(n) (+)) and the side chain are seen when beta-Caa is at the N-terminus of the peptides. MS(3) of the [M--H--C(CH(3))(3)OH](-) ion of peptides containing gamma-Caa/gammaAbu at the N-terminus gave y(n) (-) and [M--H--C(CH(3))(3)OH--CO(2)](-) ions, whereas the presence of beta-Caa at the N-terminus yielded predominantly [M--H--C(CH(3))(3)OH--HNCO](-). Thus, on the basis of our previous study and that presented here we propose that the fragmentation of these hybrid carbopeptides is highly influenced by the type of carbo amino acid present at the N-terminus.     

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.     

Differentiation of three pairs of Boc-beta,gamma- and gamma,beta-hybrid peptides by electrospray ionization tandem mass spectrometry

A new series of Boc-N-beta(3), gamma(4)-/gamma(4), beta(3)-isomeric hybrid peptides (containing repeats of beta(3)-Caa and gamma(4)-Caa's, Caa = C-linked carbo beta(3)-/gamma(4)-amino acids derived from D-xylose) have been differentiated by both positive and negative ion electrospray ionization (ESI) ion-trap and high resolution quadrupole time-of-flight/tandem mass spectrometry (Q-TOF MS/MS). MS(n) of protonated isomeric peptides and [M + H - Boc + H](+) produce characteristic fragmentation involving the peptide backbone, the Boc-group, and the side chain. The positional isomers are differentiated from one another by the presence of y(n) (+), b(n) (+), and other fragment ions of different m/z values. It is observed that the peptides with beta-Caa at the N-terminus produce extensive fragmentation, whereas gamma-Caa gave rise to much less fragmentation. Peptides with gamma-Caa at the N-terminus lose NH(3), whereas this process is absent for the carbopeptides with beta-Caa at the N-terminus. Two pairs of dipeptide diastereomers are clearly differentiated by the collision-induced dissociation (CID) of their protonated molecules. The loss of 2-methylprop-1-ene is more pronounced for Boc-NH-(R)-beta-Caa-(R)-gamma-Caa-OCH(3) (6) and Boc-NH-(R)-gamma-Caa-(R)-beta-Caa-OCH(3) (12), whereas it is insignificant or totally absent for its protonated diastereomeric pair Boc-NH-(S)-beta-Caa-(S)-gamma-Caa-OCH(3) (1) and Boc-NH-(S)-gamma-Caa-(S)-beta-Caa-OCH(3) (7). Further, ESI negative ion tandem mass spectrometry has also been found to be useful for differentiating these isomeric peptide acids. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Differentiating alpha- and beta-aspartic acids by electrospray ionization and low-energy tandem mass spectrometry

Spectra obtained by low-energy electrospray ionization tandem mass spectrometry (ESI-MS/MS) of 34 peptides containing aspartic acids at position n were studied and unambiguously differentiated. beta-Aspartic acid yields an internal rearrangement similar to that of the C-terminal rearrangements of protonated and cationized peptides. As a result of this rearrangement, two different ions containing the N- and the C-terminal ends of the original peptide are formed, namely, the bn-1 + H2O and y"l - n + 1 - 46 ions, respectively, where e is the number of amino acid residues in the peptide. The structure suggested for the y"l - n + 1 - 46 ion is identical to that proposed for the vn ions observed upon high-energy collision-induced dissociation (CID) experiments. The intensity of these ions in the low-energy MS/MS spectra is greatly influenced by the presence and position of basic amino acids within the sequences. Peptides with a basic amino acid residue at position n - 1 with respect to the beta-aspartic acid yield very intense bn-1 + H2O ions, while the y"l - n + 1 - 46 ion was observed mostly in tryptic peptides. Comparison between the high- and low-energy MS/MS spectra of several isopeptides suggests that a metastable fragmentation process is the main contributor to this rearrangement, whereas for long peptides (40 AA) CID plays a more important role. We also found that alpha-aspartic acid containing peptides yield the normal immonium ion at 88 Da, while peptides containing beta-aspartic acid yield an ion at m/z 70, and a mechanism to explain this phenomenon is proposed. Derivatizing isopeptides to form quaternary amines, and performing MS/MS on the sodium adducts of isopeptides, both improve the relative intensity of the bn + 1 + H2O ions. Based on the above findings, it was possible to determine the isomerization sites of two aged recombinant growth proteins.     

Sequence-specific fragmentation of deprotonated peptides containing H or alkyl side chains

The [M - H]- ions of a variety of di- to pentapeptides containing H or alkyl side chains have been prepared by electrospray ionization and low-energy collision-induced dissociation (CID) of the deprotonated species carried out in the interface region between the atmospheric pressure source and the quadrupole mass analyzer. Using the nomenclature applied to the fragmentation of protonated peptides, deprotonated dipeptides fragment to give a2 ions (CO2 loss) and y1 ions, where the y1 ion has two fewer hydrogens than the y"1 ions formed from protonated peptides. Deprotonated tri- and tetrapeptides fragment to give primarily y1, c1, and "b2 ions, where the "b2 ion has two fewer hydrogens than the b2 ion observed for protonated peptides. More minor yields of y2, c2, and a2 ions also are observed. The a ion formed by loss of CO2 from the [M - H]- ion shows loss of the N-terminal residue for tripeptides and sequential loss of two amino acid residues from the N-terminus for tetrapeptides. The formation of c(n) ions and the sequential loss of N-terminus residues from the [M - H - CO2]- ion serves to sequence the peptide from the N-terminus, whereas the formation of y(n) ions serves to sequence the peptide from the C-terminus. It is concluded that low-energy CID of deprotonated peptides provides as much (or more) sequence information as does CID of protonated peptides, at least for those peptides containing H or alkyl side chains. Mechanistic aspects of the fragmentation reactions observed are discussed.     

Isotope edited product ion assignment by α-N labeling of peptides with [2H3(50%)]2,4-Dinitrofluorobenzene

An isotopic modification of Sanger's method for identifying peptide N-termini has been developed to assist peptide sequencing by tandem mass spectrometry. Tryptic peptides, such as Val-His-Leu-Thr-Pro-Val-Glu-Lys, are derivatized with an equimolar mixture of 2,4-dinitrofluorobenzene and [2H3]2,4-dinitrofluorobenzene. Under optimized derivatization conditions, the alpha-amino group could be derivatized while the epsilon-amine of the lysine side chain and the imidazole of histidine remained underivatized. The alpha-dinitrophenyl modified peptides were characterized by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and liquid chromatography (LC)-ESI-MS. The [M + H]+ ions showed a doublet pattern with a delta m/z of 3 and the [M + 2H]2+ ions were recognized as doublets with a delta m/z of 1.5. MS/MS was employed where both isotopic [M + 2H]2+ ions were alternately subjected to collision-induced dissociation in the second quadrupole. Fragmentation in the ionization source generated identical product ion patterns that were observed during fragmentation in the second quadrupole. In the product ion mass spectra, the N-terminal a and b ions (no c ion observed) are doublets with a delta m/z of 3 or 1.5, while the C-terminal y and z ions (no x ion observed) are singlets appearing at identical masses. Thus, the product ions containing the N-terminus derivatized with a dinitrophenyl group are unequivocally distinguished from the product ions containing the C-terminus. The dinitrophenyl modification generally enhanced the production of a and b ions without diminishing y and z ion yields.     

Multi-stage tandem mass spectrometry of metal cationized leucine enkephalin and leucine enkephalin amide

We have examined the multi-stage collision induced dissociation (CID) of metal cationized leucine enkephalin, leucine enkephalin amide, and the N-acetylated versions of the peptides using ion trap mass spectrometry. In accord with earlier studies, the most prominent species observed during the multi-stage CID of alkali metal cationized leucine enkephalin are the [b(n) + 17 + Cat]+ ions. At higher CID stages (i.e. >MS(4)), however, dissociation of the [b2 + 17 + Cat]+ ion, a cationized dipeptide, results in the production of [a(n) -1 + Cat]+ species. The multi-stage CID of Ag+ cationized leucine enkephalin can be initiated with either the [b(n) -1 + Ag]+ or [b(n) + 17 + Ag]+ ions produced at the MS/MS stage. For the former, sequential CID stages cause, in general, the loss of CO, and then the loss of the imine of the C-terminal amino acid, to reveal the amino acid sequence. Similar to the alkali cationized species, CID of [b2 -1 + Ag]+ produces prominent [a(n) -1 + Ag]+ ions. The multi-stage CID of argentinated peptides is reminiscent of fragmentation observed for protonated peptides, in that a series of (b(n)) and (a(n)) type ions are generated in sequential CID stages. The Ag+ cation is similar to the alkali metals, however, in that the [b(n) + 17 + Ag]+ product is produced at the MS/MS and MS3 stages, and that sequential CID stages cause the elimination of amino acid residues primarily from the C-terminus. We found that N-acetylation of the peptide significantly influenced the fragmentation pathways observed, in particular by promoting the formation of more easily interpreted (in the context of unambiguous sequence determination) dissociation spectra from the [b2 + 17 + Li]+, [b2 + 17 + Na]+ and [b2 -1 + Ag]+ precursor ions. Our results suggest, therefore, that N-acetylation may improve the efficacy of multi-stage CID experiments for C-terminal peptide sequencing in the gas phase. For leucine enkephalin amide, only the multi-stage CID of the argentinated peptide allowed the complete amino acid sequence to be determined from the C-terminal side.     

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.     

Selective identification and quantitative analysis of methionine containing peptides by charge derivatization and tandem mass spectrometry

To enable the development of a tandem mass spectrometry (MS/MS) based methodology for selective protein identification and differential quantitative analysis, a novel derivatization strategy is proposed, based on the formation of a "fixed-charge" sulfonium ion on the side-chain of a methionine amino acid residue contained within a protein or peptide of interest. The gas-phase fragmentation behavior of these side chain fixed charge sulfonium ion containing peptides is observed to result in exclusive loss of the derivatized side chain and the formation of a single characteristic product ion, independently of charge state or amino acid composition. Thus, fixed charge containing peptide ions may be selectively identified from complex mixtures, for example, by selective neutral loss scan mode MS/MS methods. Further structural interrogation of identified peptide ions may be achieved by subjecting the characteristic MS/MS product ion to multistage MS/MS (MS3) in a quadrupole ion trap mass spectrometer, or by energy resolved "pseudo" MS3 in a triple quadrupole mass spectrometer. The general principles underlying this fixed charge derivatization approach are demonstrated here by MS/MS, MS3 and "pseudo" MS3 analysis of side chain fixed-charge sulfonium ion derivatives of peptides containing methionine formed by reaction with phenacylbromide. Incorporation of "light" and "heavy" isotopically encoded labels into the fixed-charge derivatives facilitates the application of this method to the quantitative analysis of differential protein expression, via measurement of the relative abundances of the neutral loss product ions generated by dissociation of the light and heavy labeled peptide ions. This approach, termed "selective extraction of labeled entities by charge derivatization and tandem mass spectrometry" (SELECT), thereby offers the potential for significantly improved sensitivity and selectivity for the identification and quantitative analysis of peptides or proteins containing selected structural features, without requirement for extensive fractionation or otherwise enrichment from a complex mixture prior to analysis.     

Dehydration versus deamination of N-terminal glutamine in collision-induced dissociation of protonated peptides

Some of the most prominent "neutral losses" in peptide ion fragmentation are the loss of ammonia and water from N-terminal glutamine. These processes are studied by electrospray ionization mass spectrometry in singly- and doubly-protonated peptide ions undergoing collision-induced dissociation in a triple quadrupole and in an ion trap instrument. For this study, four sets of peptides were synthesized: (1) QLLLPLLLK and similar peptides with K replaced by R, H, or L, and Q replaced by a number of amino acids, (2) QLnK (n = 0, 1, 3, 5, 7, 9, 11), (3) QLnR (n = 0, 1, 3, 5, 7, 9), and (4) QLn (n = 1, 2, 3, 4, 8). The results for QLLLPLLLK and QLLLPLLLR show that the singly protonated ions undergo loss of ammonia and to a smaller extent loss of water, whereas the doubly protonated ions undergo predominant loss of water. The fast fragmentation next to P (forming the y5 ion) occurs to a larger extent than the neutral losses from the singly protonated ions but much less than the water loss from the doubly protonated ions. The results from these and other peptides show that, in general, when N-terminal glutamine peptides have no "mobile protons", that is, the number of charges on the peptide is no greater than the number of basic amino acids (K, R, H), deamination is the predominant neutral loss fragmentation, but when mobile protons are present the predominant process is the loss of water. Both of these processes are faster than backbone fragmentation at the proline. These results are rationalized on the basis of resonance stabilization of the two types of five-membered ring products that would be formed in the neutral loss processes; the singly protonated ion yields the more stable neutral pyrrolidinone ring whereas the doubly protonated ion yields the protonated aminopyrroline ring (see Schemes). The generality of these trends is confirmed by analyzing an MS/MS spectra library of peptides derived from tryptic digests of yeast. In the absence of mobile protons, glutamine deamination is the most rapid neutral loss process. For peptides with mobile protons, dehydration from glutamine is far more rapid than from any other amino acid. Most strikingly, end terminal glutamine is by far the most labile source of neutral loss in excess-proton peptides, but not highly exceptional when mobile protons are not available. In addition, rates of deamination are faster in lysine versus arginine C-terminus peptides and 20 times faster in positively charged than negatively charged peptides, demonstrating that these formal neutral loss reactions are not "neutral reactions" but depend on charge state and stability.     

Fragmentation chemistry of [M + Cu]+ peptide ions containing an N-terminal arginine

[M + Cu]+ peptide ions formed by matrix-assisted laser desorption/ionization from direct desorption off a copper sample stage have sufficient internal energy to undergo metastable ion dissociation in a time-of-flight mass spectrometer. On the basis of fragmentation chemistry of peptides containing an N-terminal arginine, we propose the primary Cu+ ion binding site is the N-terminal arginine with Cu+ binding to the guanidine group of arginine and the N-terminal amine. The principal decay products of [M + Cu]+ peptide ions containing an N-terminal arginine are [a(n) + Cu - H]+ and [b(n) + Cu - H]+ fragments. We show evidence to suggest that [a(n) + Cu - H]+ fragment ions are formed by elimination of CO from [b(n) + Cu - H]+ ions and by direct backbone cleavage. We conclude that Cu+ ionizes the peptide by attaching to the N-terminal arginine residue; however, fragmentation occurs remote from the Cu+ ion attachment site involving metal ion promoted deprotonation to generate a new site of protonation. That is, the fragmentation reactions of [M + Cu]+ ions can be described in terms of a "mobile proton" model. Furthermore, proline residues that are adjacent to the N-terminal arginine do not inhibit formation of [b(n) + Cu - H]+ ion, whereas proline residues that are distant to the charge carrying arginine inhibit formation of [b(n) + Cu - H]+ ions. An unusual fragment ion, [c(n) + Cu + H]+, is also observed for peptides containing lysine, glutamine, or asparagine in close proximity to the Cu+ carrying N-terminal arginine. Mechanisms for formation of this fragment ion are also proposed.     

Electron capture dissociation mass spectrometry of peptide cations containing a lysine homologue: a mobile proton model for explaining the observation of b-type product ions

Eleven doubly protonated peptides with a residue homologous to lysine were investigated by electron capture dissociation mass spectrometry (ECD-MS). Lysine homologues provide the unique opportunity to examine the ECD fragmentation behavior by allowing us to vary the length of the lysine side chain, with minimal structural change. The lysine homologue has a primary amine side chain with a length that successively decreases by one methylene (CH(2)) unit from the --CH(2)CH(2)CH(2)CH(2)NH(2) of lysine and the accompanying decrease of its proton affinities: lysine (K), 1006.5(+/-7.2) kJ/mol; ornithine (K(*)), 1001.1(+/-6.6) kJ/mol; 2,4-diaminobutanoic acid (K(**)), 975.8(+/-7.4) kJ/mol; 2,3-diaminopropanoic acid (K(***)), 950.2(+/-7.2) kJ/mol. In general, the lysine-homologous peptides exhibited overall ECD fragmentation patterns similar to that of the lysine-containing peptides in terms of the locations, abundances, and ion types of products, such as yielding c(+) and z(+.) ions as the dominant product ions. However, a close inspection of product ion mass spectra showed that ECD-MS for the alanine-rich peptides with an ornithinyl or 2,4-diaminobutanoyl residue gave rise to b ions, while the lysinyl-residue-containing peptides did not, in most cases, produce any b ions. The peptide selectivity in the generation of b(+) ions could be understood from within the framework of the mobile proton model in ECD-MS, previously proposed by Cooper (Ref. 29). The exact mass analysis of the resultant b ions reveals that these b ions are not radical species but rather the cationic species with R-CO(+) structure (or protonated oxozalone ion), that is, b(+) ions. The absence of [M+2H](+.) species in the ECD mass spectra and the selective b(+)-ion formation are evidence that the peptides underwent H-atom loss upon electron capture, and then the resulting reduced species dissociated following typical MS/MS fragmentation pathways. This explanation was further supported by extensive b(+) ions generated in the ECD of alanine-based peptides with extended conformations.     

Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (b_n-1 + H₂O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H₂O and NH₃) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment ion in the high mass range of the MS/MS spectra. The mass difference between this signal and the protonated molecular ion corresponds to the mass of the C-terminal residue. It allowed a straightforward identification of the amino acid positioned at this extremity. It must be emphasized that a neutral residue loss can be misattributed to the formation of a y_m-1 ion, i.e., to the loss of the N-terminal residue following the a₁-y_m–1 fragmentation channel. Extreme caution must be adopted when reading the direct sequence ion on the positive ion MS/MS spectra of singly charged peptides not to mix up the attribution of the N- and C-terminal amino acids. Although such peculiar fragmentation behavior is of obvious interest for de novo peptide sequencing, it can also be exploited in proteomics, especially for studies involving digestion protocols carried out with proteolytic enzymes other than trypsin (Lys-N, Glu-C, and Asp-N) that produce arginine-containing peptides.     

Effect of phenylalanine on the fragmentation of deprotonated peptides

The fragmentation reactions of a variety of deprotonated dipeptides and tripeptides containing phenylalanine have been studied using energy-resolved collision-induced dissociation, isotopic labeling and MS/MS/MS experiments. The benzyl a-group has a substantial effect on the fragmentation reactions observed. When the phenylalanine is in the C-terminal position of dipeptides or tripeptides a major fragmentation reaction is elimination of neutral cinnamic acid to from a deprotonated amino acid amide (c1 ion) for dipeptides and a deprotonated dipeptide amide (c2 ion) for tripeptides. Fragmentation of the [M - H]- ions of tripeptides with phenylalanine in the central position also results in substantial formation of the deprotonated amide of the N-terminal amino acid residue. When the phenylalanine residue is in the N-terminal position elimination of C7H8 from the [M - H - CO2]- ion and formation of the benzyl anion become important fragmentation pathways. Sequence ions frequently observed are the y1 ions, "b2 ions and a3-Nt ions.     

Mechanisms for the proton mobility-dependent gas-phase fragmentation reactions of S-alkyl cysteine sulfoxide-containing peptide ions

Mechanisms for the gas-phase fragmentation reactions of singly and multiply protonated precursor ions of the model S-alkyl cysteine sulfoxide-containing peptides GAILCGAILK, GAILCGAILR, and VTMGHFCNFGK prepared by reaction with iodomethane, iodoacetamide, iodoacetic acid, acrylamide, or 4-vinylpyridine, followed by oxidation with hydrogen peroxide, as well as peptides obtained from an S-carboxyamidomethylated and oxidized tryptic digest of bovine serum albumin, have been examined using multistage tandem mass spectrometry, hydrogen/deuterium exchange and molecular orbital calculations (at the B3LYP/6-31 + G(d,p) level of theory). Consistent with previous reports, CID-MS/MS of the S-alkyl cysteine sulfoxide-containing peptide ions resulted in the dominant "non-sequence" neutral loss of an alkyl sulfenic acid (XSOH) from the modified cysteine side chains under conditions of low proton mobility, irrespective of the alkylating reagent employed. Dissociation of uniformly deuterated precursor ions of these model peptides determined that the loss of alkyl sulfenic acid in each case occurred via a "charge-remote" five-centered cis-1,2 elimination reaction to yield a dehydroalanine-containing product ion. Similarly, the charge state dependence to the mechanisms and product ion structures for the losses of CO(2), CO(2) + H(2)O and CO(2) + CH(2)O from S-carboxymethyl cysteine sulfoxide-containing peptides, and for the losses of CH(2)CHCONH(2) and CH(2)CHC(5)H(4)N, respectively, from S-amidoethyl and S-pyridylethyl cysteine sulfoxide-containing peptide ions have also been determined. The results from these studies indicate that both the proton mobility of the peptide precursor ion and the nature of the S-alkyl substituent have a significant influence on the abundances and charge states of the product ions resulting from the various competing fragmentation pathways.     

Role of the sulfhydryl group on the gas phase fragmentation reactions of protonated cysteine and cysteine containing peptides

The gas phase fragmentation reactions of protonated cysteine and cysteine-containing peptides have been studied using a combination of collisional activation in a tandem mass spectrometer and ab initio calculations [at the MP2(FC)/6-31G*//HF/6-31G* level of theory]. There are two major competing dissociation pathways for protonated cysteine involving: (i) loss of ammonia, and (ii) loss of the elements of [CH₂O₂]. MS/MS, MS/MS of selected ions formed by collisional activation in the electrospray ionization source as well as ab initio calculations have been carried out to determine the mechanisms of these reactions. The ab initio results reveal that the most stable [M + H − NH₃]⁺ isomer is an episulfonium ion (A), whereas the most stable [M + H − CH₂O₂]⁺ isomer is an immonium ion (B). The effect of the position of the cysteine residue on the fragmentation reactions of the [M + H]⁺ ions of all the possible simple dipeptide and tripeptide methyl esters containing one cysteine (where all other residues are glycine) has also been investigated. When cysteine is at the N-terminal position, NH₃ loss is observed, although the relative abundance of the resultant [M + H − NH₃]⁺ ion decreases with increasing peptide size. In contrast, when cysteine is at any other position, water loss is observed. The proposed mechanism for loss of H₂O is in competition with those channels leading to the formation of structurally relevant sequence ions.     

Characterization of Nα-Fmoc-protected dipeptide isomers by electrospray ionization tandem mass spectrometry (ESI-MS(n)): effect of protecting group on fragmentation of dipeptides

A series of positional isomeric pairs of Fmoc-protected dipeptides, Fmoc-Gly-Xxx-OY/Fmoc-Xxx-Gly-OY (Xxx=Ala, Val, Leu, Phe) and Fmoc-Ala-Xxx-OY/Fmoc-Xxx-Ala-OY (Xxx=Leu, Phe) (Fmoc=[(9-fluorenylmethyl)oxy]carbonyl) and Y=CH(3)/H), have been characterized and differentiated by both positive and negative ion electrospray ionization ion-trap tandem mass spectrometry (ESI-IT-MS(n)). In contrast to the behavior of reported unprotected dipeptide isomers which mainly produce y(1)(+) and/or a(1)(+) ions, the protonated Fmoc-Xxx-Gly-OY, Fmoc-Ala-Xxx-OY and Fmoc-Xxx-Ala-OY yield significant b(1)(+) ions. These ions are formed, presumably with stable protonated aziridinone structures. However, the peptides with Gly- at the N-terminus do not form b(1)(+) ions. The [M+H](+) ions of all the peptides undergo a McLafferty-type rearrangement followed by loss of CO(2) to form [M+H-Fmoc+H](+). The MS(3) collision-induced dissociation (CID) of these ions helps distinguish the pairs of isomeric dipeptides studied in this work. Further, negative ion MS(3) CID has also been found to be useful for differentiating these isomeric peptide acids. The MS(3) of [M-H-Fmoc+H](-) of isomeric peptide acids produce c(1)(-), z(1)(-) and y(1)(-) ions. Thus the present study of Fmoc-protected peptides provides additional information on mass spectral characterization of the dipeptides and distinguishes the positional isomers.     

Electrospray ionization tandem mass spectrometry of model peptides reveals diagnostic fragment ions for protein ubiquitination

In this work, synthetic peptides were used to determine the fragmentation behavior of ubiquitinated peptides and to find ions diagnostic for peptide ubiquitination. The ubiquitin-calmodulin peptide1 was chosen as the model peptide for naturally occurring ubiquitinated proteins cleaved with endoproteinase gluC. In addition, the fragmentation behavior of model ubiquitinated peptides produced by tryptic digestion was also of great interest since the standard protocols for proteomics-based protein identification use trypsin as the protease. Attachment of ubiquitin to a target protein results in a branched structure, but only ions from the ubiquitin side chain (and the lysine to which it is attached) can be used as diagnostic ions, since fragment ions that contain other amino acids from the parent protein will vary in mass. Characteristic b-type fragment ions from the gluC cleavage of the ubiquitin side chain (designated as b ions) were found which involve only the ubiquitin tail (b2, b3, b4, b5 and b6 ions at m/z 189.06, 302.12, 439.18, 552.30 and 651.30, respectively). Maximum production of these ions occurred at a collision energy of 45 eV in a Q-TOF instrument. Although a non-ubiquitinated peptide may produce isobaric fragment ions, it is unlikely that it can produce these ions in combination. With liquid chromatography/tandem mass spectrometry (LC/MS/MS) experiments, ubiquitinated peptides can readily be determined by surveying the reconstructed or extracted ion chromatograms of the diagnostic fragment ions for common peaks. Characteristic ions resulting from tryptic cleavage of the side chain were found in cleavage products with a missed cleavage, resulting in a LRGG- tag instead of a GG- tag. For the LRGG-tagged peptide, diagnostic MS/MS fragment ions (at m/z 270.17 and 384.21) from the ubiquitin tail (b2 and b4, respectively) were found, along with an internal fragment ion (LRGGK-28) at m/z 484.30. These ions should prove useful in precursor-ion scanning experiments for identifying peptides modified by attachment of ubiquitin, and for locating the site of ubiquitin attachment.