Fragmentation of the deprotonated ions of peptides containing cysteine, cysteine sulfinic acid, cysteine sulfonic acid, aspartic acid, and glutamic acid

We examined the fragmentation of the electrospray-produced [M-H]- and [M-2H]2- ions of a number of peptides containing two acidic amino acid residues, one being aspartic acid (Asp) or glutamic acid (Glu), and the other being cysteine sulfinic acid [C(SO2H)] or cysteine sulfonic acid [C(SO3H)], on an ion-trap mass spectrometer. We observed facile neutral losses of H2S and H2SO2 from the side chains of cysteine and C(SO2H), respectively, whereas the corresponding elimination of H2SO3 from the side chain of C(SO3H) was undetectable for most peptides that we investigated. In addition, the collisional activation of the [M-H]- ions of the C(SO2H)-containing peptides resulted in the cleavage of the amide bond on the C-terminal side of the C(SO2H) residue. Moreover, collisional activation of the [M-2H]2- ions of the above Asp-containing peptides led to the cleavage of the backbone N-Calpha bond of the Asp residue to give cn and/or its complementary [zn-H2O] ions. Similar cleavage also occurred for the singly deprotonated ions of the otherwise identical peptides with a C-terminal amide functionality, but not for the [M-H]- ions of same peptides with a free C-terminal carboxylic acid. Furthermore, ab initio calculation results for model cleavage reactions are consistent with the selective cleavage of the backbone N-Calpha bond in the Asp residue.     

Fragmentation of protonated ions of peptides containing cysteine,cysteine sulfinic acid,and cysteine sulfonic acid

The oxidation of the sulfhydryl group in cysteine to sulfenic acid, sulfinic acid, and sulfonic acid in proteins is important in a number of enzymatic processes. In this study we examined the fragmentation of four peptides containing cysteine, cysteine sulfinic acid (Cys-SO(2)H), and cysteine sulfonic acid (Cys-SO(3)H) in an ion-trap mass spectrometer. Our results show that the presence of a Cys-SO(2)H in a peptide leads to preferential cleavage of the amide bond at the C-terminal side of the oxidized cysteine residue. The results are important for the determination of the site of the cysteine oxidation and might be useful for the sequencing of cysteine-containing peptides.     

Mobile and localized protons: a framework for understanding peptide dissociation

Protein identification and peptide sequencing by tandem mass spectrometry requires knowledge of how peptides fragment in the gas phase, specifically which bonds are broken and where the charge(s) resides in the products. For many peptides, cleavage at the amide bonds dominate, producing a series of ions that are designated b and y. For other peptides, enhanced cleavage occurs at just one or two amino acid residues. Surface-induced dissociation, along with gas-phase collision-induced dissociation performed under a variety of conditions, has been used to refine the general 'mobile proton' model and to determine how and why enhanced cleavages occur at aspartic acid residues and protonated histidine residues. Enhanced cleavage at acidic residues occurs when the charge is unavailable to the peptide backbone or the acidic side-chain. The acidic H of the side-chain then serves to initiate cleavage at the amide bond immediately C-terminal to Asp (or Glu), producing an anhydride. In contrast, enhanced cleavage occurs at His when the His side-chain is protonated, turning His into a weak acid that can initiate backbone cleavage by transferring a proton to the backbone. This allows the nucleophilic nitrogen of the His side-chain to attack and form a cyclic structure that is different from the 'typical' backbone cleavage structures.     

Gln-Gly cleavage: a dominant dissociation site in the fragmentation of protonated peptides

An understanding of the gas-phase dissociation of protonated peptides within the mass spectrometer is essential for automated high-throughput protein identification. In this communication we describe a facile cleavage of the Gln-Gly peptide bond under low-collisional energy conditions. A variety of synthetic peptides have been analysed where key amino acids have been substituted within the sequence PQGPPQQGGR, which is a consensus repeat present in the tryptic peptides of acidic proline-rich protein 1 (PRP-1). The collision-induced dissociation spectra obtained from the PRP-1 tryptic peptides and the synthetic peptides indicate that facile Gln-Gly cleavage occurs when an X-Gln-Gly-Y sequence is present in a peptide, where X is any amino acid and Y any amino acid other than Gly.     

Dissociation of the peptide bond in protonated peptides

The dissociation of the amide (peptide) bond in protonated peptides, [M + H](+), is discussed in terms of the structures and energetics of the resulting N-terminal b(n) and C-terminal y(n) sequence ions. The combined data provide strong evidence that dissociation proceeds with no reverse barriers through interconverting proton-bound complexes between the segments emerging upon cleavage of the protonated peptide bond. These complexes contain the C-terminal part as a smaller linear peptide (amino acid if one residue) and the N-terminal part either as an oxazolone or a cyclic peptide (cyclic amide if one residue). Owing to the higher thermodynamic stability but substantially lower gas-phase basicity of cyclic peptides vs isomeric oxazolones, the N-terminus is cleaved as a protonated oxazolone when ionic (b(n) series) but as a cyclic peptide when neutral (accompanying the C-terminal y(n) series). It is demonstrated that free energy correlations can be used to derive thermochemical data about sequence ions. In this context, the dependence of the logarithm of the abundance ratio log[y(1)/b(2)], from protonated GGX (G, glycine; X, varying amino acid) on the gas-phase basicity of X is used to obtain a first experimental estimate of the gas-phase basicity of the simplest b-type oxazolone, viz. 2-aminomethyl-5-oxazolone (b(2) ion with two glycyl residues).     

Collision-Induced Dissociation Fragmentation Inside Disulfide C-Terminal Loops of Natural Non-Tryptic Peptides

Collision-induced dissociation (CID) spectra of long non-tryptic peptides are usually quite complicated and rather difficult to interpret. Disulfide bond formed by two cysteine residues at C-terminus of frog skin peptides precludes one to determine sequence inside the forming loop. Thereby, chemical modification of S–S bonds is often used in “bottom up” sequencing approach. However, low-energy CID spectra of natural non-tryptic peptides with C-terminal disulfide cycle demonstrate an unusual fragmentation route, which may be used to elucidate the “hidden” C-terminal sequence. Low charge state protonated molecules experience peptide bond cleavage at the N-terminus of C-terminal cysteine. The forming isomeric acyclic ions serve as precursors for a series of b-type ions revealing sequence inside former disulfide cycle. The reaction is preferable for peptides with basic lysine residues inside the cycle. It may also be activated by acidic protons of Asp and Glu residues neighboring the loop. The observed cleavages may be quite competitive, revealing the sequence inside disulfide cycle, although S–S bond rupture does not occur in this case.

Computational investigation and hydrogen/deuterium exchange of the fixed charge derivative tris(2,4,6-Trimethoxyphenyl) phosphonium: Implications for the aspartic acid cleavage mechanism

Aspartic acid (Asp)-containing peptides with the fixed charge derivative tris(2,4,6trimethoxyphenyl) phosphonium (tTMP-P+) were explored computationally and experimentally by hydrogen/deuterium (H/D) exchange and by fragmentation studies to probe the phenomenon of selective cleavage C-terminal to Asp in the absence of a "mobile" proton. Ab initio modeling of the tTMP-P+ electrostatic potential shows that the positive charge is distributed on the phosphonium group and therefore is not initiating or directing fragmentation as would a "mobile" proton. Geometry optimizations and vibrational analyses of different Asp conformations show that the Asp structure with a hydrogen bond between the side-chain hydroxy and backbone carbonyl lies 2.8 kcal/mol above the lowest energy conformer. In reactions with D2O, the phosphonium-derived doubly charged peptide (H+)P+LDIFSDF rapidly exchanges all 12 of its exchangeable hydrogens for deuterium and also displays a nonexchanging population. With no added proton, P+LDIFSDF exchanges a maximum of 4 of 11 exchangeable hydrogens for deuterium. No exchange is observed when all acidic groups are converted to the corresponding methyl esters. Together, these H/D exchange results indicate that the acidic hydrogens are "mobile locally" because they are able to participate in exchange even in the absence of an added proton. Fragmentation of two distinct (H+)P+LDIFSDF ion populations shows that the nonexchanging population displays selective cleavage, whereas the exchanging population fragments more evenly across the peptide backbone. This result indicates that H/D exchange can sometimes distinguish between and provide a means of separation of different protonation motifs and that these protonation motifs can have an effect on the fragmentation.     

Computational study on nonenzymatic peptide bond cleavage at asparagine and aspartic acid

Nonenzymatic peptide bond cleavage at asparagine (Asn) and glutamine (Gln) residues has been observed during peptide deamidation experiments; cleavage has also been reported at aspartic acid (Asp) and glutamic acid (Glu) residues. Although peptide backbone cleavage at Asn is known to be slower than deamidation, fragmentation products are often observed during peptide deamidation experiments. In this study, mechanisms leading to the cleavage of the carboxyl-side peptide bond of Asn and Asp residues were investigated using computational methods (B3LYP/6-31+G**). Single-point solvent calculations at the B3LYP/6-31++G** level were carried out in water, utilizing the integral equation formalism-polarizable continuum (IEF-PCM) model. Mechanism and energetics of peptide fragmentation at Asn were comparatively analyzed with previous calculations on deamidation of Asn. When deamidation proceeds through direct hydrolysis of the Asn side chain or through cyclic imide formationvia a tautomerization routeit exhibits lower activation barriers than peptide bond cleavage at Asn. The fundamental distinction between the mechanisms leading to deamidationvia a succinimideand backbone cleavage was found to be the difference in nucleophilic entities involved in the cyclization process (backbone versus side-chain amide nitrogen). If deamidation is prevented by protein three-dimensional structure, cleavage may become a competing pathway. Fragmentation of the peptide backbone at Asp was also computationally studied to understand the likelihood of Asn deamidation preceding backbone cleavage. The activation barrier for backbone cleavage at Asp residues is much lower (approximately 10 kcal/mol) than that at Asn. This suggests that peptide bond cleavage at Asn residues is more likely to take place after it has deamidated into Asp.     

Chemical Mimics of Aspartate-Directed Proteases: Predictive and Strictly Specific Hydrolysis of a Globular Protein at Asp−X Sequence Promoted by Polyoxometalate Complexes Rationalized by a Combined Experimental and Theoretical Approach

Creating efficient and residue-directed artificial proteases is a challenging task due to the extreme inertness of the peptide bond, combined with the difficulty of achieving specific interactions between the catalysts and the protein side chains. Herein we report strictly site-selective hydrolysis of a multi-subunit globular protein, hemoglobin (Hb) from bovine blood, by a range of Zr^IV-substituted polyoxometalates (Zr-POMs) in mildly acidic and physiological pH solutions. Among 570 peptide bonds in Hb, selective cleavage was observed at only eleven sites, each occurring at Asp−X peptide bonds located in the positive patches on the protein surface. The molecular origins of the observed Asp−X selectivity were rationalized by means of molecular docking, DFT-based binding, and mechanistic studies on model peptides. The proposed mechanism of hydrolysis involves coordination of the amide oxygen to Zr^IV followed by a direct nucleophilic attack of the side chain carboxylate group on the C-terminal amide carbon atom with formation of a cyclic anhydride, which is further hydrolyzed to give the reaction products. The activation energy for the cleavage of the structurally related Glu−X sequence compared to Asp−X was calculated to be higher by 1.4 kcal mol⁻¹, which corresponds to a difference of about one order of magnitude in the rates of hydrolysis. The higher activation energy is attributed to the higher strain present in the six-membered ring of glutaric anhydride (Glu−X), as compared to the five-membered ring of the succinic anhydride (Asp−X) intermediate. Similarly, the cleavage at X−Asp and X−Glu bonds are predicted to be kinetically less likely as the corresponding activation energies were 6 kcal mol⁻¹ higher_, explaining the experimentally observed selectivity. The synergy between the negatively charged polyoxometalate cluster, which binds at positive patches on protein surfaces, and selective activation of Asp−X peptide bonds located in these regions by Zr^IV ions, results in a novel class of artificial proteases with aspartate-directed reactivity, which is very rare among naturally occurring proteases.     

Aspartic acid side chain effect—Experimental and theoretical insight

Gas-phase H/D exchange and density functional theory study of the Asp and Glu side-chain carboxylic group intrinsic reactivity is reported. H/D exchange site specific treatment and some additional theoretical calculations showed that a side-chain carboxylic group may initiate proton transfer along with bond formation to one of its oxygens, i.e., possibility to initiate selective of cleavage peptide bond ("aspartic acid effect"). That finding is used to select aspartic acid cleavage mechanisms (side-chain proton transfer either to backbone carbonyl or to amide nitrogen) for further computational study. B3LYP/6-31G(d) and G3(MP2)//B3LYP potential energy profiles of both mechanisms on a model system CH3CO-Asp-NHCH3 were constructed. Although energy employed in low-energy collision induced dissociation suffices for both mechanisms thresholds, energy transferred to specific modes suggests a complex one-step mechanism of proton transfer (from the side-chain carboxylic group to the backbone amide group), bond formation (between deprotonated carboxylic group and carbon atom of the backbone carbonyl), and peptide bond cleavage as favorable.     

Spectroscopic study on interaction of nucleic acid base with tryptophan-containing tripeptides: acetyl-Trp-X-Trp-NHCH3 (X = Gly, Asn, Asp, Gln and Glu).

As part of a series of peptides designed to have binding ability selective for each of the nucleic acid bases, five tripeptides consisting of N-acetyl-Trp-X-Trp-NHCH3 (X = Gly, Asn, Asp, Gln and Glu) were synthesized, and their abilities to form complexes with four different nucleotides were examined by the fluorescence and phase distribution methods. The association constants obtained indicated that, depending on the sort of X residue, the peptides showed a variation in their interaction with guanosine monophosphate (GMP), while no noticeable selectivity was observed for other nucleotides adenosine monophosphate (AMP), uridine monophosphate (UMP) and cytidine monophosphate (CMP). The binding mode of N-acetyl-Trp-Asp-Trp-NHCH3 for the guanine base was further investigated using the proton nuclear magnetic resonance (1H-NMR) method. The mode was suggested to involve intimate cooperation of (1) the hydrogen bond formation between the carboxyl group of the Asp side chain and the guanine C2-amino group, and (2) the stacking interaction of the base with two terminal Trp residues of the peptide. Such interaction was strengthened by the protonation of the guanine base. A tentative binding mode is proposed based on these results.     

Characterization by tandem mass spectrometry of stable cysteine sulfenic acid in a cysteine switch peptide of matrix metalloproteinases

Cysteine sulfenic acid (Cys-SOH) is an elusive intermediate in reactive oxygen species-induced oxidation reactions of many proteins such as peroxiredoxins and tyrosine phosphatases. Cys-SOH is proposed to play a vital role in catalytic and signaling functions. The formation of cysteine sulfinic acid (Cys-SO(2)H) and cysteine sulfonic acid (Cys-SO(3)H) has been implicated in the activation of matrix metalloproteinase-7 (MMP-7) and oxidation of thiol to cysteine sulfinic acid has been associated with the autolytic cleavage of MMP-7. We have examined the formation of cysteine sulfenic acid in a synthetic peptide PRCGVPDVA, which is a cysteine switch domain of MMP-7 and other matrix metalloproteases. We have prepared the cysteine sulfenic acid containing peptide, PRC(SOH)GVPDVA, by reaction with hydroxyl radicals generated by the Fenton reaction (Fe(+2)/H(2)O(2)). We characterized this modified peptide by tandem mass spectrometry and accurate mass measurement experiments. In addition, we used 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl) reagent to form an adduct with PRC(SOH)GVPDVA to provide additional evidence for the viability of PRC(SOH)GVPDVA in solution. We also characterized an intramolecular cysteine sulfinamide cross-link product PRC[S(O)N]GVPDVA based on tandem mass spectrometry and accurate mass measurement experiments. These results contribute to the understanding of a proteolytic cleavage mechanism that is traditionally associated with MMP activation.     

Identification of degradation products of aspartyl tripeptides by capillary electrophoresis-tandem mass spectrometry

Capillary electrophoresis-electrospray tandem mass spectrometry (CE-MS/MS) has been used to identify degradation products of the aspartyl tripeptides Phe-Asp-GlyNH(2) and Gly-Asp-PheNH(2) following incubation of the peptides in acidic and alkaline solution. At pH 2, the dominant decomposition products resulted from cleavage of the peptide backbone amide bonds to yield the respective dipeptides and amino acids. In addition, the cyclic aspartyl succinimide intermediate was identified by its [M+H](+) at m/z = 319 and the MS/MS spectrum exhibiting a simple fragmentation pattern with the [C(8)H(10)N](+)-ion as the principal daughter ion (a(1) of Phe-Asp-GlyNH(2)). Deamidation of the C-terminal amide as well as isomerization and enantiomerization of the Asp residue occurred upon incubation at pH 10. alpha-Asp and the isomeric beta-Asp and most of the diastereomeric forms (corresponding to D/L-Asp) could be separated by CE. All isomers could be identified based on their MS/MS spectra. Peptides with the amino acid sequence Phe-Asp-Gly containing the regular alpha-Asp bond displayed a highly intense b(2) fragment ion and a low abundant y(2) ion. In contrast, the y(2) and a(1) fragment were high abundant daughter ions in the mass spectra of beta-Asp peptides while the b(2) ion exhibited a lower abundance. Differences in the MS/MS spectra of the isomers of the peptides with the sequence Gly-Asp-Phe were obvious but less pronounced. In conclusion, CE-MS/MS proved to be a useful tool to study the decomposition and enantiomerization of peptides including the isomerization of Asp residues, due to the combination of efficient separation of isomers by CE and their identification by MS/MS.     

Atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry of sulfonic acid derivatized tryptic peptides.

Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) and ion trap mass spectrometry have been used to study the fragmentation behavior of native peptides and peptide derivatives prepared for de novo sequencing applications. Sulfonic acid derivatized peptides were observed to fragment more extensively and up to 28 times more efficiently than the corresponding native peptides. Tandem mass spectra of native peptides containing aspartic or glutamic acids are dominated by cleavage on the C-terminal side of the acidic residues. This significantly limits the amount of sequence information that can be derived from those compounds. The MS/MS spectra of native tryptic peptides containing oxidized Met residues show extensive loss of CH(3)SOH and little sequence-specific fragmentation. On the other hand, the tandem mass spectra of derivatized peptides containing Asp, Glu and oxidized Met show much more uniform fragmentation along the peptide backbone. The AP-MALDI tandem mass spectra of some derivatized peptides were shown to be qualitatively very similar to the corresponding vacuum MALDI postsource decay mass spectra, which were obtained on a reflector time-of-flight instrument. However, the ion trap mass spectrometer offers several advantages for peptide sequencing relative to current reflector time-of-flight instruments including improved product ion mass measurement accuracy, improved precursor ion selection and MS(n). These latter capabilities were demonstrated with solution digests of model proteins and with in-gel digests of 2D-gel separated proteins.     

Palladium(II) complex as a sequence-specific peptidase: hydrolytic cleavage under mild conditions of X-Pro peptide bonds in X-Pro-Met and X-Pro-His segments

The X-Pro peptide bond (in which X represents any amino acid residue) in peptides and proteins is resistant to cleavage by most proteolytic enzymes. We show that [Pd(H(2)O)(4)](2+) ion can selectively hydrolyze this tertiary peptide bond within the X-Pro-Met and X-Pro-His sequence segments. The hydrolysis requires an equimolar amount of the Pd(II) reagent and occurs under mild conditions-at temperature as low as 20 degrees C (with half-life of 1.0 h at pH 2.0) and at pH as high as 7.0 (with half-life of 4.2 h at pH 7.0 and 40 degrees C). The secondary peptide bond, exemplified by X-Gly in the X-Gly-Met and X-Gly-His sequence segments, however, is cleaved only in weakly acidic solution (pH < 4.0) and more slowly (half-life is 4.2 h at pH 2.0 and 60 degrees C). We explain the sequence-specificity of X-Pro cleavage by NMR spectroscopic analysis of the coordination of the X-Pro-Met segment to the Pd(II) ion. We give indirect evidence for the mechanism of cleavage by analyzing the conformation of the scissile X-Pro peptide bond, and by comparing the rate constants for the cleavage of the tertiary X-Pro peptide bond, the tertiary X-Sar peptide bond (Sar is N-methyl glycine), and the typical secondary X-Gly peptide bond in a set of analogous oligopeptides. Methionine and histidine side chains provide the recognition by selectively binding (anchoring) the Pd(II) ion. The proline residue provides the enhanced activity because its tertiary X-Pro peptide bond favors the cleavage-enhancing binding of the Pd(II) ion to the peptide oxygen atom and prevents the cleavage-inhibiting binding of the Pd(II) ion upstream of the anchoring (histidine or methionine) residue. Cleavage can be switched from the residue-selective to the sequence-specific mode by simply adjusting the pH of the aqueous solution. In acidic solutions, any X-Y bond in X-Y-Met and X-Y-His segments is cleaved because the cleavage is directed by anchoring methionine and histidine residues. In mildly acidic and neutral solutions, only the X-Pro bond in X-Pro-Met and X-Pro-His sequences is cleaved because of an interplay between the anchoring residue and the proline residue preceding it. Because Pro-Met and Pro-His sequences are rare in proteins, this sequence-specific cleavage is potentially useful for the removal of the fusion tags from the bioengineered fusion proteins.     

Negative ion fragmentations of deprotonated peptides: backbone cleavages directed through both Asp and Glu

The collision-induced spectra of [M - H](-) ions of a variety of natural and synthetic amphibian peptides containing Asp and/or Glu exhibit characteristic gamma backbone cleavage ions that identify the positions of these residues in the peptide. A theoretical study suggests that the Glu cleavage involves an S(N)i reaction of the carboxylate anion from the Glu alpha side chain to form a deprotonated cyclic lactone. The presence of either Asp or Glu or other residues that effect pronounced side-chain cleavages (e.g. Ser or Thr) results in the normal alpha and beta backbone cleavages being reduced in comparison to those cleavages which originate from side chains.     

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.     

Cascade Dissociations of Peptide Cation-Radicals. Part 1. Scope and Effects of Amino Acid Residues in Penta-, Nona-, and Decapeptides

Amino acid residue-specific backbone and side-chain dissociations of peptide z ions in MS(3) spectra were elucidated for over 40 pentapeptides with arginine C-terminated sequences of the AAXAR and AAHXR type, nonapeptides of the AAHAAXX"AR and AAHAXAX"AR type, and AAHAAXX"AAR decapeptides. Peptide z(n) ions containing amino acid residues with readily transferrable benzylic or tertiary β-hydrogen atoms (Phe, Tyr, His, Trp, Val) underwent facile backbone cleavages to form dominant z(n-2) or z(n-3) ions. These backbone cleavages are thought to be triggered by a side-chain β-hydrogen atom transfer to the z ion C(α) radical site followed by homolytic dissociation of the adjacent C(α)-CO bond, forming x(n-2) cation-radicals that spontaneously dissociate by loss of HNCO. Amino acid residues that do not have readily transferrable β-hydrogen atoms (Gly, Ala) do not undergo the z(n) → z(n-2) dissociations. The backbone cleavages compete with side-chain dissociations in z ions containing Asp and Asn residues. Side-chain dissociations are thought to be triggered by α-hydrogen atom transfers that activate the C(β)-C(γ) or C(β)-heteroatom bonds for dissociations that dominate the MS(3) spectra of z ions from peptides containing Leu, Cys, Lys, Met, Ser, Arg, Glu, and Gln residues. The Lys, Arg, Gln, and Glu residues also participate in γ-hydrogen atom transfers that trigger other side-chain dissociations.     

Impact of proline and aspartic acid residues on the dissociation of intermolecularly crosslinked peptides

The dissociation of intermolecularly crosslinked peptides was evaluated for a series of peptides with proline or aspartic acid residues positioned adjacent to the crosslinking sites (lysine residues). The peptides were crosslinked with either disuccinimidyl suberate (DSS) or disuccinimidyl L-tartrate (DST), and the influence of proline and aspartic acid residues on the fragmentation patterns were investigated for precursor ions with and without a mobile proton. Collisionally activated dissociation (CAD) spectra of aspartic acid-containing crosslinked peptide ions, doubly-charged with both protons sequestered, were dominated by cleavage C-terminal to the Asp residue, similar to that of unmodified peptides. The proline-containing crosslinked peptides exhibited a high degree of internal ion formation, with the resulting product ions having an N-terminal proline residue. Upon dissociation of the doubly-charged crosslinked peptides, twenty to fifty percent of the fragment ion abundance was accounted for by multiple cleavage products. Crosslinked peptides possessing a mobile proton yielded almost a full series of b- and y-type fragment ions, with only proline-directed fragments still observed at high abundances. Interestingly, the crosslinked peptides exhibited a tendency to dissociate at the amide bond C-terminal to the crosslinked lysine residue, relative to the N-terminal side. One could envision updating computer algorithms to include these crosslinker specific product ions--particularly for precursor ions with localized protons--that provide complementary and confirmatory information, to offer more confident identification of both the crosslinked peptides and the location of the crosslink, as well as affording predictive guidelines for interpretation of the product-ion spectra of crosslinked peptides.     

Investigation of the neutral loss of a full amino acid mass during collision-induced dissociation of the b(3)+ ion derived from a model peptide containing a 4-aminobutyric acid residue

In a previous study we found that a dominant fragmentation pathway observed for collision-induced dissociation (CID) of b(3)+ derived from peptides with sequence AXAG, where X is gamma-aminobutyric acid (gammaAbu) or epsilon-aminocaproic acid (Cap), involved the loss of 89 mass units (u). A neutral loss of 89 u corresponded to the free acid mass of an alanine (A) residue. This specific pathway was studied in greater detail here using a series of A(gammaAbu)AG peptides with strategic positioning of (15)N, (13)C and (2)H isotope labels. Based on the extensive labeling, several possible routes to the net elimination of 89 u are proposed. One is based on initial elimination of either aziridinone or imine and CO, followed by opening of an oxazolinone, tautomerization and elimination of H2O. Another involves formation of an aziridinone by cleavage of the N-terminal amide bond, and transfer of O and H atoms to this fragment via an H-bonded ion-molecule complex to complete the loss of 89 u. Both types of pathway include the transfer/migration of H atoms from the alpha-carbon position of gammaAbu or A residues.