Fragmentation reactions of protonated peptides containing glutamine or glutamic acid

A variety of protonated dipeptides and tripeptides containing glutamic acid or glutamine were prepared by electrospray ionization or by fast atom bombardment ionization and their fragmentation pathways elucidated using metastable ion studies, energy-resolved mass spectrometry and triple-stage mass spectrometry (MS(3)) experiments. Additional mechanistic information was obtained by exchanging the labile hydrogens for deuterium. Protonated H-Gln-Gly-OH fragments by loss of NH(3) and loss of H(2)O in metastable ion fragmentation; under collision-induced dissociation (CID) conditions loss of H-Gly-OH + CO from the [MH - NH(3)](+) ion forms the base peak C(4)H(6)NO(+) (m/z 84). Protonated dipeptides with an alpha-linkage, H-Glu-Xxx-OH, are characterized by elimination of H(2)O and by elimination of H-Xxx-OH plus CO to form the glutamic acid immonium ion of m/z 102. By contrast, protonated dipeptides with a gamma-linkage, H-Glu(Xxx-OH)-OH, do not show elimination of H(2)O or formation of m/z 102 but rather show elimination of NH(3), particularly in metastable ion fragmentation, and elimination of H-Xxx-OH to form m/z 130. Both the alpha- and gamma-dipeptides show formation of [H-Xxx-OH]H(+), with this reaction channel increasing in importance as the proton affinity (PA) of H-Xxx-OH increases. The characteristic loss of H(2)O and formation of m/z 102 are observed for the protonated alpha-tripeptide H-Glu-Gly-Phe-OH whereas the protonated gamma-tripeptide H-Glu(Gly-Gly-OH)-OH shows loss of NH(3) and formation of m/z 130 as observed for dipeptides with the gamma-linkage. Both tripeptides show abundant formation of the y(2)' ion under CID conditions, presumably because a stable anhydride neutral structure can be formed. Under metastable ion conditions protonated dipeptides of structure H-Xxx-Glu-OH show abundant elimination of H(2)O whereas those of structure H-Xxx-Gln-OH show abundant elimination of NH(3). The importance of these reaction channels is much reduced under CID conditions, the major fragmentation mode being cleavage of the amide bond to form either the a(1) ion or the y(1)' ion. Particularly when Xxx = Gly, under CID conditions the initial loss of NH(3) from the glutamine containing dipeptide is followed by elimination of a second NH(3) while the initial loss of H(2)O from the glutamic acid dipeptide is followed by elimination of NH(3). Isotopic labelling shows that predominantly labile hydrogens are lost in both steps. Although both [H-Gly-Glu-Gly-OH]H(+) and [H-Gly-Gln-Gly-OH]H(+) fragment mainly to form b(2) and a(2) ions, the latter also shows elimination of NH(3) plus a glycine residue and formation of protonated glycinamide. Isotopic labelling shows extensive mixing of labile and carbon-bonded hydrogens in the formation of protonated glycinamide.     

Photodissociation and spectroscopic study of cold protonated dipeptides

A photodissociation spectrometer, containing a spray ionization source and a temperature-variable multipole ion trap, has been constructed to examine the structure and reactivity of gas phase biological molecular ions at various temperatures. Ultraviolet (UV) and infrared (IR) photodissociation spectra of protonated alanyltryptophan (Ala-TrpH+) and tryptophanylglycine (Trp-GlyH+) have been measured. In UV spectra, the S1-S0 band origin of Ala-TrpH+ exhibits a significant red shift with respect to those of protonated tryptophan (TrpH+) and Trp-GlyH+. This red shift is ascribed to the stabilization of the excited state due to the strong interaction between the NH3+ group and indole ring. We also discuss the temperature effect on the structure and reactivity for these peptides. In addition to the UV photodissociation spectra of the dipeptides, IR spectra of the complex of Ala-TrpH+ with methanol are measured. IR photodissociation spectra of solvated ions show that Ala-TrpH+-methanol has the closed structure, which is consistent with the large spectral shift in UV spectrum of bare dipeptide.     

Reaction competition in the fragmentation of protonated dipeptides

A major low-energy fragmentation reaction of many protonated dipeptides involves cleavage of the amide bond resulting in formation of either the y(1)" ion or the a(1) ion. For a series of protonated dipeptides H-Val-Xxx-OH it is observed that log(y(1)"/a(1)) is a linear function of the proton affinity of the variable C-terminal amino acid. For the series of protonated dipeptides H-Xxx-Phe-OH log(a(1)/y(1)") gives a poor correlation with the proton affinity or gas-phase basicity of H-Xxx-OH. However, a good limited correlation of log(a(1)/y(1)") with the Taft-Topsom sigma(alpha) for the alkyl group is observed when Xxx is an aliphatic amino acid. It is proposed that fragmentation occurs by initial formation of a proton-bound complex of an aziridinone and an amino acid which may fragment to form either a protonated amino acid (y(1)") or an N-protonated aziridinone with the corresponding neutrals being an aziridinone and an amino acid. Ab initio calculations show that the N-protonated aziridinone is unstable and fragments by loss of CO to form the a(1) immonium ion. However, the proton-bound complex of an aziridinone and an amine base is a stable species which exists in a potential well. Copyright 2000 John Wiley & Sons, Ltd.     

Fragmentation cross sections of protonated water clusters

We have measured fragmentation cross sections of protonated water cluster cations (H(2)O)(n=30-50)H(+) by collision with water molecules. The clusters have well-defined sizes and internal energies. The collision energy has been varied from 0.5 to 300 eV. We also performed the same measurements on deuterated water clusters (D(2)O)(n=5-45)D(+) colliding with deuterated water molecules. The main fragmentation channel is shown to be a sequential thermal evaporation of single molecules following an initial transfer of relative kinetic energy into internal energy of the cluster. Unexpectedly, that initial transfer is very low on average, of the order of 1% of collision energy. We evaluate that for direct collisions (i.e., within the hard sphere radius), the probability for observing no fragmentation at all is more than 35%, independently of cluster size and collision energy, over our range of study. Such an effect is well known at higher energies, where it is attributed to electronic effects, but has been reported only in a theoretical study of the collision of helium atoms with sodium clusters in that energy range, where only vibrational excitation occurs.     

Low-energy tautomers and conformers of neutral and protonated arginine.

The relative stabilities of zwitterionic and canonical forms of neutral arginine and of its protonated derivative were studied by using ab initio electronic structure methods. Trial structures were first identified at the PM3 level of theory with use of a genetic algorithm to systematically vary geometrical parameters. Further geometry optimizations of these structures were performed at the MP2 and B3LYP levels of theory with basis sets of the 6-31++G** quality. The final energies were determined at the CCSD/6-31++G** level and corrected for thermal effects determined at the B3LYP level. Two new nonzwitterionic structures of the neutral were identified, and one of them is the lowest energy structure found so far. The five lowest energy structures of neutral arginine are all nonzwitterionic in nature and are clustered within a narrow energy range of 2.3 kcal/mol. The lowest energy zwitterion structure is less stable than the lowest nonzwitterion structure by 4.0 kcal/mol. For no level of theory is a zwitterion structure suggested to be the global minimum. The calculated proton affinity of 256.3 kcal/mol and gas-phase basicity of 247.8 kcal/mol of arginine are in reasonable agreement with the measured values of 251.2 and 240.6 kcal/mol, respectively. The calculated vibrational characteristics of the low-energy structures of neutral arginine provide an alternative interpretation of the IR-CRLAS spectrum (Chapo et al. J. Am. Chem. Soc. 1998, 120, 12956-12957).     

Conformation-specific infrared and ultraviolet spectroscopy of tyrosine-based protonated dipeptides

We present the spectroscopy and photofragmentation dynamics of two isomeric protonated dipeptides, H+AlaTyr and H+TyrAla, in a cold ion trap. By a combination of infrared-ultraviolet double resonance experiments and density functional theory calculations, we establish the conformations present at low temperature. Interaction of the charge at the N-terminus with the carbonyl group and the tyrosine pi-cloud seems to be critical in stabilizing the low-energy conformations. H+AlaTyr has the flexibility to allow a stronger interaction between the charge and the aromatic ring than in H+TyrAla, and this interaction may be responsible for many of the differences we observe in the former: a significant redshift in the ultraviolet spectrum, a much larger photofragmentation yield, fewer stable conformations, and the absence of fragmentation in excited electronic states.     

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.     

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.     

Modeling of the gas-phase ion chemistry of protonated arginine

Arginine is often involved at the C-terminus of peptides obtained from tryptic digests of proteins. The very basic guanidine group of the side-chain of arginine has a large effect on the backbone fragmentation of protonated peptides. Furthermore, arginine exhibits specific fragmentation reactions involving its side-chain. Various tautomerization states, conformers and side-chain dissociation channels of protonated arginine were studied using theoretical methods. The guanidine loss of protonated arginine is proved to be an S(N)2 substitution on the delta-carbon of the side-chain, starting from species containing the N(epsilon)H-C(+)(N(eta)H(2))(N(eta')H(2)) or -N(epsilon) (+)H(2)-C(N(eta)H)(N(eta')H(2)) moieties and leads to formation to either protonated guanidine or protonated proline. In the corresponding transition structures the proline moiety is protonated. Under low-energy collision conditions the extra proton transfers to the guanidine moiety, leading to the formation of C(+)(NH(2))(3). On the other hand, the lifetime of the fragmenting species under high-energy collision conditions is shorter, resulting in enhanced formation of protonated proline and its dissociation products. The first step of ammonia loss is the leaving of a preformed NH(3) from tautomers containing the -N(epsilon)H-C(N(eta)H(3) (+))(N(eta')H) or -N(epsilon)-C(N(eta)H(3) (+))(N(eta')H(2)) moieties. The resulting protonated carbodiimide group can be stabilized by intramolecular nucleophilic attack, leading to ring formation. Overall, reactions involved in the ammonia loss from protonated arginine can be considered as an S(N)1 substitution on the central zeta-carbon of the guanidine group.     

Fragmentation of deprotonated cyclic dipeptides by electrospray ionization mass spectrometry

The fragmentation pathways of deprotonated cyclic dipeptides have been studied by electrospray ionization multi‐stage mass spectrometry (ESI‐MSⁿ) in negative mode. The results showed that the fragmentation pathways of deprotonated cyclic dipeptides depended significantly on the different substituents, the side chains of amino acid residues at the diketopiperazine ring. In the spectra of deprotonated cyclic dipeptides, the ion [M? H? substituent radical]^? was firstly observed in the ESI mode. The characteristic fragment ions [M? H? substituent radical]^? and [M? H? (substituent? H)]^? could be used as the symbols of particular cyclic dipeptides. The hydrogen/deuterium (H/D) exchange experiment, the high‐resolution mass spectrometry (Q‐TOF) and theoretical calculations were used to rationalize the proposed fragmentation pathways and to verify the differences between the fragmentation pathways. The relative Gibbs free energies (ΔG) of the product ions and possible fragmentation pathways were estimated using the B3LYP/6–31++G(d, p) model. The results have some potential applications in the structural elucidation and interpretation of the mass spectra of homologous compounds and will enrich the gas‐phase ESI‐MS ion chemistry of cyclic dipeptides. Copyright © 2009 John Wiley & Sons, Ltd.     

Fragmentation of protonated oligoalanines: Amide bond cleavage and beyond

The fragmentation reactions of the singly-protonated oligoalanines trialanine to hexaalanine have been studied using energy-resolved mass spectrometry in MS(2) and MS(3) experiments. The primary fragmentation reactions are rationalized in terms of the b(x)-y(z) pathway of amide bond cleavage which results in formation of a proton-bound complex of an oxazolone and a peptide/amino acid; on decomposition of this complex the species of higher proton affinity preferentially retains the proton. For protonated pentaalanine and protonated hexaalanine the major primary fragmentation reaction involves cleavage of the C-terminal amide bond to form the appropriate b ion. The lower mass b ions originate largely, if not completely, by further fragmentation of the initially formed b ion. MS(3) energy-resolved experiments clearly show the fragmentation sequence b(n) --> b(n-1) --> b(n-2). A more minor pathway for the alanines involves the sequence b(n) --> a(n) --> b(n-1) --> b(n-2). The a(5) ion formed from hexaalanine loses, in part, NH(3) to begin the sequence of fragmentation reactions a(5) --> a(5)* --> a(4)* --> a(3)* where a(n)* = a(n) - NH(3). The a(3)* ion also is formed from the b(3) ion by the sequence b(3) --> a(3) --> a(3)* with the final step being sufficiently facile that the a(3) ion is not observed with significant intensity in CID mass spectra. A cyclic structure is proposed for the a(3)* ion.     

Energy dependence of rearrangements of an N-terminal protecting group in tandem mass spectrometry of protonated tripeptides containing C-terminal arginine

The types and intensities of tandem mass spectrometric products of side-chain interactions were investigated with a hybrid tandem instrument. Positive-ion unimolecular decomposition and collisionally activated decomposition studies were conducted on the [M + H]⁺ ions of two N-benzyloxycarbonyl (Cbz or Z)-protected tripeptides, Cbz-Gly-Leu-Arg-NH₂ and Cbz-Gly-Pro-Arg-NH₂. The loss of benzyl alcohol (108 u) and the formation of other significant product ions and their dependence on collision energy and gas pressure suggest reaction between both ends of the molecule. Replacement of leucine with proline at the second position in the tripeptide produces a very intense [M + H ? 108]⁺ ion and fewer lower mass fragment ions in the tandem mass spectra for Cbz-Gly-Pro-Arg-NH₂ than in those for Cbz-Gly-Leu-Arg-NH₂.     

Gas phase hydrogen/deuterium exchange of arginine and arginine dipeptides complexed with alkali metals

The hydrogen/deuterium (H/D) exchange of protonated and alkali-metal cationized Arg-Gly and Gly-Arg peptides with D(2)O in the gas phase was studied using electrospray ionization quadropole ion trap mass spectrometry. The Arg-Gly and Gly-Arg alkali metal complexes exchange significantly more hydrogens than protonated Arg-Gly and Gly-Arg. We propose a mechanism where the peptide shifts between a zwitterionic salt bridge and nonzwitterionic charge solvated conformations. The increased rate of H/D exchange of the alkali metal complexes is attributed to the peptide metal complexes' small energy difference between the salt-bridge conformation and the nonzwitterionic charge-solvated conformation. Implications for the applicability of this mechanism to other zwitterionic systems are discussed.     

IR-LD spectroscopic characterization of L-Tryptophan containing dipeptides

IR-spectroscopic and stereo-structural analysis of aromatic l-Tryptophan containing dipeptides l-Tryptophan-l-Tryptophan (Trp-Trp), l-Tyrosine-l-Tryptophan (Tyr-Trp) and cyclo(Trp-Trp) have been carried out by means of solid-state linear-dichroic infrared (IR-LD) spectroscopy of oriented as suspension in nematic liquid crystal solids. The experimental data have been compared with analogous ones of the simple amino acids l-Tyrosnine (l-Tyr) and l-Tryptophan (l-Trp). In cyclo(Trp-Trp), the IR-spectral changes towards the IR-ones of acyclic Trp-Trp have been determined. A theoretical analysis of Tyr-Trp at Hartree-Fock level of theory and 6-31G** basis set is also applied.     

Fragmentation of protonated thioether conjugates of acrolein using low collision energies

The protonated mercapturic acid conjugate of acrolein, S-(3-oxopropyl)-N-acetyl-L-cysteine (I), undergoes facile retro-Michael loss of acrolein in the gas phase. To determine whether extensive loss of acrolein would impede structural characterization of acrolein-peptide adducts, fragmentation reactions of a series of conjugates, formed by 1,4-Michael addition of acrolein to peptides and cysteine derivatives, were investigated at collision cell potentials up to ?50 V using a triple quadrupole mass spectrometer. Differences in fragmentation dynamics suggest protonation at the sulfur of the N-acetylcysteine conjugate I facilitates retro-Michael elimination of acrolein with a low activation energy relative to other fragmentations. Analogous fragmentation was eliminated after borohydride reduction of the aldehyde to an alcohol. Retro-Michael fragmentation was not significant for acrolein conjugates of glutathione derivatives, suggesting that proton sequestration occurs in peptides with multiple amide linkages even when the peptide does not contain a basic amino group. An unexpected outcome of these experiments was the observation of a facile gas-phase cleavage of peptides on the N-terminal side of S-(3-oxopropyl)cysteine residues. Such fragmentation behavior may prove useful for locating cysteine residues in peptides.     

The bimolecular hydrogen-deuterium exchange behavior of protonated alkyl dipeptides in the gas phase

As part of an ongoing characterization of the intrinsic chemical properties of peptides, thermal hydrogen-deuterium exchange has been studied for a series of fast-atom-bombardment-generated protonated alkyldipeptides and related model compounds in the reaction with D₂O, CH₃OD, and ND₃ in a Fourier transform ion cyclotron resonance mass spectrometer. Despite the very large basicity difference between the dipeptides and the D₂O and CH₃OD exchange reagents, efficient exchange of all active hydrogen atoms occurs. From the kinetic data it appears that exchange of the amino, amide, and hydroxyl hydrogens proceeds with different efficiencies, which implies that the proton in thermal protonated dipeptides is immobile. The selectivity of the exchange at the different basic sites is governed by the nature of both the dipeptide and the exchange reagent. The results indicate that reversible proton transfer in the reaction complexes, which effectuates the deuterium incorporation, is assisted by formation of multiple hydrogen bonds between the reagents. Exchange is considered to proceed via the intermediacy of different competing intermediate complexes, each of which specifically leads to deuterium incorporation at different basic sites. The relative stabilization of the competing intermediate complexes can be related to the relative efficiencies of deuterium incorporation at different basic sites in the dipeptide. For all protonated dipeptides studied, the exchange in the reaction with ND₃ proceeds with unit efficiency, whereas all active hydrogen atoms are exchanged equally efficiently. Evidently specific multiple hydrogen bond formations are far less important in the reversible proton transfers with the relatively basic ammonia, which allows effective randomization of all active hydrogen atoms in the reaction complexes.     

Competing gas-phase fragmentation pathways of asparagine-, glutamine-, and lysine-containing protonated dipeptides

The fragmentation chemistry of protonated H–Val–Asn–OH, H–Val–Gln–OH and H–Val–Lys–OH is investigated in this work by means of modeling and density functional theory calculations. Former experimental studies indicate that the ratio of a ₁ and y ₁ ions cannot be explained by considering the proton affinities of the corresponding dissociating species on the a ₁–y ₁ pathway, while the fragmentation of other dipeptides can be understood in this way. We demonstrate that considering the correct PA value for H–Asn–OH eliminates the deviation observed for H–Val–Asn–OH. The larger than expected a ₁/y ₁ ratio of H–Val–Gln–OH is explained by considering the dissociation kinetics of the proton-bound dimers formed on the a ₁–y ₁ pathway and competition of the deamidation and a ₁–y ₁ channels. For H–Val–Lys–OH, it is proposed that a ₁ ions are indeed formed from one of the primary products, protonated H–Val–Cap–OH.     

Fragmentation of protonated amides through intermediate ion-neutral complexes: Neighboring group participation

It has been shown that neighboring group participation plays an important role in the fragmentation of protonated amides; the attachment of an adjacent functional group capable of accepting a proton provides alternative pathways of low energy for the formation of the inevitable N-protonated species in the fragmentation of the amide bond. Under methane chemical ionization (CI) conditions, protonated aniline (m/z 94) is only 1. 6% of the base peak MH⁺ ion for acetanilide; the abundance of the m/z 94 ion is increased to 15% for acetoacetanilide and protonated o-methoxyaniline reaches a relative intensity of 49% for N-acetyl-o-methoxyaniline. A more striking difference in ease of the formation of protonated anilines is found for acetanilides bearing a nitro group at different positions. Protonated nitroaniline (m/z 139) is the base peak in the methane CI spectrum of N-acetyl-o-nitroaniline; the m/z 139 ion drops to only 0.7% for the para isomer, and this ion is increased to 31.5% in the spectrum of N-acetoaceto-p-nitroaniline. By employing low energy collision-induced dissociation, it has been found that the fragmentation of protonated amides proceeds by way of ion-neutral complexes. In the case of acetanilide, for example, the cleavage of the amide bond gives rise to an acetylium ion and neutral aniline, which are bound together as a complex. An α-hydrogen of the acetylium ion, which is activated by the positive charge, is captured by aniline due to its higher proton affinity as compared with ketene. For those compounds having mobile protons other than the amidic hydrogen, it is indicated that such proton has the priority to be transferred in the reaction. Thus, the proton on the free carboxyl group of N-phenyl succinic and maleic monoamides is transferred in the fragmentation, leading to anhydrides as the neutral species in the formation of protonated aniline.