Further studies on the fragmentation of protonated ions of peptides containing aspartic acid, glutamic acid, cysteine sulfinic acid, and cysteine sulfonic acid

Here we examined the fragmentation, on a quadrupole ion-trap mass spectrometer, of the protonated ions of a group of peptides containing one arginine and two different acidic amino acids, one being aspartic acid (Asp) or glutamic acid (Glu) and the other being cysteine sulfinic acid [C(SO2H)] or cysteine sulfonic acid [C(SO3H)]. Our results showed that, upon collisional activation, the cleavage of the peptide bond C-terminal to C(SO2H) is much more facile than that of the peptide bond C-terminal to Asp, Glu, or C(SO3H). There is no significant difference, however, in susceptibility to cleavage of peptide bonds that are C-terminal to Asp, Glu, and C(SO3H). To understand these experimental observations, we carried out B3LYP/6-31G* density functional theory calculations for a model cleavage reaction of GXG --> b2 + Gly, in which X is Asp, Glu, C(SO2H), or C(SO3H). Our calculation results showed that the cleavage reaction is thermodynamically more favorable when X = C(SO2H) than when X = Asp or C(SO3H). We attributed the less facile cleavage of the amide bond after Glu to that the formation of a six-membered ring b ion for Glu-bearing peptides is kinetically not as favorable as the formation of a five-membered ring b ion for peptides containing the other three acidic amino acids. The results from this study may provide useful tools for peptide sequencing.     

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.     

Improving peptide fragmentation by N-terminal derivatization with high proton affinity

An improved method of de novo peptide sequencing based on mass spectrometry using novel N-terminal derivatization reagents with high proton affinity has been developed. The introduction of a positively charged group into the N-terminal amino group of a peptide is known to enhance the relative intensity of b-ions in product ion spectra, allowing the easy interpretation of the spectra. However, the physicochemical properties of charge derivatization reagents required for efficient fragmentation remain unclear. In this study, we prepared several derivatization reagents with high proton affinity, which are thought to be appropriate for peptide fragmentation under low-energy collision-induced dissociation (CID) conditions, and examined their usefulness in de novo peptide sequencing. Comparison of the effects on fragmentation among three derivatization reagents having a guanidino or an amidino moiety, which differ in proton affinity, clearly indicated that there was an optimal proton affinity for efficient fragmentation of peptides. Among reagents tested in this study, derivatization with 4-amidinobenzoic acid brought about the most effective fragmentation. This derivatization approach will offer a novel de novo peptide sequencing method under low-energy CID conditions.     

Tandem mass spectrometry of peptides with N-terminal glutamine studies on a prion protein peptide

Journal of The American Society for Mass Spectrometry - This paper reports the unusual collision-induced fragmentation of peptides having N-terminal glutamine. One of these glutamine-containing...     

Structure and fragmentation of b2 ions in peptide mass spectra

In a number of cases the b2 ion observed in peptide mass spectra fragments directly to the a1 ion. The present study examines the scope of this reaction and provides evidence as to the structure(s) of the b2 ions undergoing fragmentation to the a1 ion. The b2 ion H-Ala-Gly+ fragments, in part, to the a1 ion, whereas the isomeric b2 ion H-Gly-Ala+ does not fragment to the a1 ion. Ab initio calculations of ion energies show that this different behavior can be rationalized in terms of protonated oxazolone structures for the b2 ions provided one assumes a reverse activation energy of approximately 1 eV for the reaction b2-->a2; such a reverse activation energy is consistent with experimental kinetic energy release measurements. Experimentally, the H-Aib-Ala+ b2 ion, which must have a protonated oxazolone structure, fragments extensively to the a1 ion. We conclude that the proposal by Eckart et al. (J. Am. Soc. Mass Spectrom. 1998, 9, 1002) that the b2 ions which undergo fragmentation to a1 ions have an immonium ion structure is not necessary to rationalize the results, but that the fragmentation does occur from a protonated oxazolone structure. It is shown that the b2-->a1 reaction occurs extensively when the C-terminus residue in the b2 ion is Gly and with less facility when the C-terminus residue is Ala. When the C-terminus residue is Val or larger, the b2-->a1 reaction cannot compete with the b2-->a2 fragmentation reaction. Some preliminary results on the fragmentation of a2 ions are reported.     

C-terminal amino acid residue loss for deprotonated peptide ions containing glutamic acid, aspartic acid, or serine residues at the C-terminus

Deprotonated peptides containing C-terminal glutamic acid, aspartic acid, or serine residues were studied by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer with ion production by electrospray ionization (ESI). Additional studies were performed by post source decay (PSD) in a matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometer. This work included both model peptides synthesized in our laboratory and bioactive peptides with more complex sequences. During SORI-CID and PSD, [M - H]- and [M - 2H]2- underwent an unusual cleavage corresponding to the elimination of the C-terminal residue. Two mechanisms are proposed to occur. They involve nucleophilic attack on the carbonyl carbon of the adjacent residue by either the carboxylate group of the C-terminus or the side chain carboxylate group of C-terminal glutamic acid and aspartic acid residues. To confirm the proposed mechanisms, AAAAAD was labelled by 18O specifically on the side chain of the aspartic acid residue. For peptides that contain multiple C-terminal glutamic acid residues, each of these residues can be sequentially eliminated from the deprotonated ions; a driving force may be the formation of a very stable pyroglutamatic acid neutral. For peptides with multiple aspartic acid residues at the C-terminus, aspartic acid residue loss is not sequential. For peptides with multiple serine residues at the C-terminus, C-terminal residue loss is sequential; however, abundant loss of other neutral molecules also occurs. In addition, the presence of basic residues (arginine or lysine) in the sequence has no effect on C-terminal residue elimination in the negative ion mode.     

Integrating Lys-N proteolysis and N-terminal guanidination for improved fragmentation and relative quantification of singly-charged ions

Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA The study of isolated protein complexes has greatly benefited from recent advances in mass spectrometry instrumentation and quantitative, isotope labeling techniques. The comprehensive characterization of protein complex components and quantification of their relative abundance relies heavily upon maximizing protein and peptide sequence information obtained from MS and tandem MS studies. Recent work has shown that using a metalloendopeptidase, Lys-N, for proteomic analysis of biological protein mixtures produces complementary protein sequence information compared with trypsin digestion alone. Here, we have investigated the suitability of Lys-N proteolysis for use with MALDI mass spectrometry to characterize the yeast Arp2 complex and E. coli PAP I protein interactions. Although Lys-N digestion resulted in an average decrease in protein sequence coverage of ∼30% compared with trypsin digestion, CID analysis of singly-charged Lys-N peptides yielded a more extensive b-ions series compared with complementary tryptic peptides. Taking advantage of this improved fragmentation pattern, we utilized differential ¹⁵N/¹⁴N guanidination of Lys-N peptides and MALDI-MS/MS analysis to relatively quantify the changes in PAP I associations due to deletion of sprE, previously shown to regulate PAP I-dependent polyadenylation. Overall, this Lys-N/guanidination integrative approach is applicable for functional proteomic studies utilizing MALDI mass spectrometry analysis, as it provides an effective and economical mean for relative quantification of proteins in conjunction with increased sensitivity of detection and fragmentation efficiency.     

Stability and visible absorption of glutamic acid complexes with uranyl and neodymium ions

Summary The stability constants of the complexes of uranyl and neodymium ions with glutamic acid are determined pH-metrically in 0.1 M NaClO₄ solution at 25° C. In both cases protonated complexes are formed in significant concentrations. A new MA complex is found in the uranyl glutamic acid system. In accordance with this investigation a graphical treatment of the visible spectral data gives the molar absorption coefficients of both MA and MHA species.

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Untersuchung der Stabilität und der Absorption im Sichtbaren von Uranyl- und Neodymkomplexen von Glutaminsäure

Zusammenfassung Die Stabilitätskonstanten der Uranylund Neodymkomplexe von Glutaminsäure wurden pH-metrisch in 0,1 M NaClO₄-Lösung bei 25° C bestimmt. In beiden Fällen werden in beträchtlichen Konzentrationen protonierte Komplexe gebildet. Ein neuer MA-Komplex wurde im Uranyl-Glutaminsäure-System gefunden. Eine graphische Behandlung der spektralen Daten im sichtbaren Bereich ergab die molaren Absorptionskoeffizienten für die MA- und MHA-Komplexe.

     

Individual b2 ion fragmentation profiles combined with AspN digestion improve N-terminal peptide sequencing

The N terminus of peptides generated by AspN is restricted to about 40 dipeptide motifs starting with D or E. These motifs are visible upon collision-induced dissociation (CID) as b₂ ions, which are often the most abundant low-mass fragment ions. It was observed that b₂ ions are accompanied by a set of sequence-specific neutral losses of CO, H₂O, NH₃, and some other small units. To test the utility of these profiles as additional parameters for reliable assignment of the b₂ ion motif besides its m/z value, the CID spectra of 221 different AspN-generated peptides covering all N-terminal D-X and E-X motifs were recorded. Qualitatively, the b₂ ion fragmentation profiles of individual motifs were found to exhibit little dependency on the rest of the peptide sequence. Thus, it is concluded that the set of b₂ ion fragmentation profiles recorded in this study can be used as reference set. Knowledge of these profiles provides an increased specificity for b₂ ion annotation of AspN-generated peptides compared to the use of only a solitary b₂ ion m/z value. Recognition of the b₂ ion motif provides a two-amino-acid sequence including its direction; it provides the location of this motif at the N terminus, and it sets a starting point for further extension of the b ion series.     

A peptide containing aspartic acid, glutamic acid and serine in calf brain synaptic vesicles

Free amino acids and other amino compounds in calf brain synaptic vesicles were identified and determined by thin-layer chromatography and ion-exchange chromatography. The vesicles contained ten identified amino acids with glutamic acid, aspartic acid, taurine and gamma-aminobutyric acid in the highest concentrations, and also cysteic acid (or cysteinesulfinic acid), glutamine, alanine, serine, glycine and lysine. The vesicles also contained certain unknown acid-labile, ninhydrin-positive compounds, one of which was a peptide yielding, after acid hydrolysis, about 40% aspartic acid, 30% serine, 15% glutamic acid, 10% glycine and possibly some alanine and lysine. The concentration of the peptide in the vesicles was as high as that of all the other amino compounds together.     

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.     

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.     

Rapid determination of underivatized pyroglutamic acid, glutamic acid, glutamine and other relevant amino acids in fermentation media by LC-MS-MS

Determination of amino acids in a complex matrix without derivatization is advantageous, however, difficulties are found in both the detection and the separation of those compounds. In this study, a rapid and reliable LC-MS-MS method for the quantitation of underivatized amino acids in exocellular media was established. Injections were made directly after centrifugation of the samples, without further preparation. The separation of seven underivatized amino acids was achieved on a reversed-phase C18 column with pentadecafluorooctanoic acid as a volatile ion-pair reagent, and the specific detection of most amino acids was achieved by MS-MS of the specific transitions [M + H]+-->[M + H - 46]+. The calibration curves of all analytes were linear over the range of 1.0-1000 microg ml(-1) and the detection limits ranged from 0.1 to 5 ng ml(-1), with an injection volume of 20 microl. The inter-day and intra-day precisions ranged from 2.6 to 5.7% and 4.8 to 8.2%, respectively; the mean recoveries of the seven analytes were 81-104%, 91-107% and 93-101% respectively at the spiked level of 10, 40 and 200 microg ml(-1). A large number of fermentation samples were analysed using this method. The technique is simple, rapid, selective and sensitive, and shows potential for the high-throughput quantitation of amino acids from other biological matrices.     

Leaving group effects on the selectivity of the gas-phase fragmentation reactions of side chain fixed-charge-containing peptide ions

The effect of trialkylsulfonium versus quaternaryalkylammonium ions on the multistage gas-phase fragmentation reactions of side chain, fixed-charge, cysteine-containing peptides has been examined in a quadrupole linear ion trap. These tandem mass spectrometry experiments reveal that selective loss of dialkylsulfide from fixed-charge sulfonium ion derivatives is the dominant fragmentation pathway regardless of the degree of proton mobility, indicating that it is the most energetically favored fragmentation pathway. In contrast, the loss of trimethylamine from the side chain of fixed-charge ammonium-ion-containing cysteine derivatives appears to be less energetically favored, and as a result extensive charge-remote fragmentation is observed under low proton mobility conditions, while under conditions of high proton mobility, amide bond fragmentation reactions dominate. These findings are supported by molecular orbital calculations at the B3LYP/6-31 + G(d, p) level of theory, which showed that the neutral loss of dimethylsulfide is both thermodynamically and kinetically preferred over the loss of trimethylamine.     

The structure and fragmentation of B n (n≥3) ions in peptide spectra

The unimolecular and low energy collision-induced fragmentation reactions of the MH⁺ ions of N-acetyl-tri-alanine, N-acetyl-tri-alanine methyl ester, N-acetyl-tetra-alanine, tetra-alanine, penta-alanine, hexa-glycine, and Leu-enkephalin have been studied with a particular emphasis on the formation and fragmentation of B _n (n=3,4,5) ions. In addition, the metastable ion fragmentation reactions of protonated tetra-glycine, penta-glycine, and Leu-enkephalin amide have been studied. B _n ions are prominent stable species in all spectra. The B _n ions fragment, in part, by elimination of CO to form A _n ions; this reaction occurs on the metastable ion time scale with a substantial release of kinetic energy (T _1/2=0. 3–0. 5 eV) that indicates that a stable configuration of the B _n ion fragments by way of a reacting configuration that is higher in energy than the fragmentation products, A _n + CO. Ab initio calculations strongly suggest that the stable configuration of the B₃ and B₄ ions is a protonated oxazolone formed by interaction of the developing charge with the next-nearest carbonyl group as HX is lost from the protonated species H-(Yyy) _n -X · H⁺. The higher B _n ions also fragment, in part, to form the next-lower B ion, presumably in its stable protonated oxazolone form. This reaction is rationalized in terms of the three-dimensional structure of the B _n ions and it is proposed that the neutral eliminated is an α-lactam.     

Synthesis of glutamic acid and glutamine peptides possessing a trifluoromethyl ketone group as SARS-CoV 3CL protease inhibitors

Trifluoromethyl-β-amino alcohol 11 [(4S)-tert-butyl 4-amino-6,6,6-trifluoro-5-hydroxyhexanoate] was synthesized in five steps starting from Cbz-l-Glu-OH 5 where the key step involved the introduction of the trifluoromethyl (CF₃) group to oxazolidinone 7, resulting in the formation of silyl ether 8 [(4S,5S)-benzyl 4-(2-(tert-butoxycarbonyl)ethyl)-5-(trifluoromethyl)-5-(trimethylsilyloxy)oxazolidine-3-carboxylate]. Compound 11 was then converted into four tri- and tetra-glutamic acid and glutamine peptides (1-4) possessing a CF₃-ketone group that exhibited inhibitory activity against severe acute respiratory syndrome coronavirus protease (SARS-CoV 3CL^pro).     

Use of reversed-phase high-performance liquid chromatography for simultaneous determination of glutamine synthetase and glutamic acid decarboxylase in crude extracts

Glutamine and gamma-aminobutyric acid (GABA), formed from glutamic acid in crude tissue extracts by glutamine synthetase and glutamic acid decarboxylase respectively, were separated by derivatization with dansyl chloride followed by reversed-phase high-performance liquid chromatography on the Altex Ultrasphere ODS-5 column. The mobile phase was a gradient of 100 mM potassium dihydrogen phosphate (pH 2.1) with 0-40% acetonitrile. The amounts of glutamine and GABA formed from glutamic acid were determined under different reaction conditions.