Fast atom bombardment mass spectrometric study of benzyloxycarbonyl-protected tetrapeptides containing proline

Fragmentations of N-benzyloxycarbonyl-protected tetrapeptide ethyl esters containing L -alanine and L -proline, in which changes in the numbers and positions of prolyl residues were observed, were studied by negative-ion fast atom bombardment mass spectrometry. A significant difference was found among the abundances of the molecule ions and the fragment ions formed by the cleavage of the benzyloxycarbonyl group, depending on the numbers and positions of prolyl residues in the derivatives. The results indicate that the conformational differences in the tetrapeptide derivatives due to the existence of proline affect fragmentations of molecules in the solution phase in negative-ion fast atom bombardment mass spectrometry.     

Fragmentations of peptide derivatives depending on the positions of proline in negative-ion fast atom bombardment mass spectrometry

Fragmentations of N-benzyloxycarbonyl-protected tripeptide ethyl esters, in which changes in the numbers and positions of prolyl residues were observed, were examined by negative-ion fast atom bombardment mass spectrometry. A significant difference was found among the intensities of the fragment ions formed by cleavage of the benzyloxycarbonyl group, depending on the numbers and positions of prolyl residues in the derivatives. These results suggest that the fragmentations of the peptide derivatives in negative-ion fast atom bombardment mass spectrometry depend on the conformational differences in the peptide derivatives due to the existence of proline, and permit the prediction of the positions of proline in the peptides.     

Mass spectrometry of 2-substituted-1-keto-3-aryl-5H-imidazo-(1,5-a)-pyrroles derived from Schiff bases of N-terminal prolyl peptides

We have been able to extend the use of Schiff base derivatives in peptide sequencing to N-terminal prolyl peptides. Earlier studies from this laboratory revealed that certain aromatic Schiff bases of peptide esters gave electron-impact mass spectra with relatively intense molecular, sequence and internal fragment ions. We observed that the reaction of N-terminal prolyl peptide esters with 4-dimethylaminonaphthaldehyde, p-dimethylaminobenzaldehyde and 2-pyridinecarboxaldehyde gave cyclization products which were found to be 2-substituted-1-keto-3-aryl-5H-imidazo-[1,5-a]-pyrrole derivatives. The molecular ion and many of the expected cleavages were prominent in the mass spectra. Deuterium labeling at the α-carbon, amide nitrogen, or other exchangeable positions has been used in assigning the structure. It was also confirmed by the fragmentation pattern of the products derived by permethylation of the peptide derivative with tetramethylammonium hydroxide. Comparable cleavage patterns were seen among the N-terminal prolyl peptides examined. Proline amide gave the corresponding cyclized product. With the inclusion of N-terminal prolyl peptides in the list of peptides that we have examined, we may now prepare volatile derivatives of peptides containing any of the protein amino acids in two steps: esterification and treatment with the appropriate aromatic aldehyde.     

Photoionisation mass spectra of volatile derivatives of short peptides and appearance potentials of their characteristic ions

Mass spectra of the volatile derivatives of short peptides were studied by the photoionisation method with the use of monochromatic photons. The dependence of the intensity of the main peaks on the photon energy was analysed from 7·5 to 13·0 eV. The data obtain reveal the influence of the chemical structure of amino acid residues on the relative probability of the decomposition of peptide molecular ions at the CH? CO and CO? NH bonds, resulting in the formation of positively charged aldimine and amino acid N-terminal fragments, respectively. These data may be used as a basis for the application of the photoionisation technique to mass spectrometric sequential studies in peptides. In peptides containing residues of aliphatic amino acid the decomposition results mainly in the formation of aldimine ions, the stability of which increase with the increase of the alkyl chain size. In peptides containing residues of aromatic amino acids the decomposition is usually observed leading to formation of the amino acid ions. Ionisation potentials, as well as photoionisation efficiency curves and appearance potentials were determined for characteristic ions. Comparison was made of the values of the appearance potentials of different fragments formed upon decomposition of molecular ions. It has been shown that for peptides containing aliphatic amino acid moieties the appearance potentials of aldimine fragments are always lower than those inherent to peptides containing aromatic amino acid residues. The larger the size of an aliphatic chain, the lower is the energy of formation of these fragments. For all the compounds studied, including the peptides containing aromatic amino acid residues, the appearance potentials of the aldimine ions did not exceed those of the amino acid ions. These data indicate that, contrary to the experiments with electron-impact with energy of about 70 eV, upon ionisation with photons with energy from 7·5 to 13·0 eV, the aldimine fragments appear directly due to primary decomposition of molecular ions, independent of the formation of the amino acid fragments. The photoionisation efficiency curves for peptides containing different types of amino acid residues facilitate the choice of an optimal photon energy providing unequivocal information on the amino acid sequence in the peptide under study.     

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

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

The mass spectra of N-adamantoylpeptides

The N-adamantoyl derivatives of the esters of twenty-one di-peptides and eight tri-peptides containing the amino acids glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, glutamic acid, lysine, histidine, serine, threonine, methionine, S-benzylcysteine and tyrosine were prepared and their mass spectra determined. The spectra of all compounds were suitable for amino acid sequence determination. Mixtures containing N-adamantoyl dipeptide and tripeptide methyl esters were separated by thin-layer chromatography and the components identified by high resolution mass spectrometry. The mass spectrometric features of N-adamantoyl peptide esters are discussed and compared with those of other N-acyl peptide derivatives.     

The dehydroalanine effect in the fragmentation of ions derived from polypeptides

The fragmentation of peptides and proteins upon collision‐induced dissociation (CID) is highly dependent on sequence and ion type (e.g. protonated, deprotonated, sodiated, odd electron, etc.). Some amino acids, for example aspartic acid and proline, have been found to enhance certain cleavages along the backbone. Here, we show that peptides and proteins containing dehydroalanine, a non‐proteinogenic amino acid with an unsaturated side‐chain, undergo enhanced cleavage of the N—Cα bond of the dehydroalanine residue to generate c‐ and z‐ions. Because these fragment ion types are not commonly observed upon activation of positively charged even‐electron species, they can be used to identify dehydroalanine residues and localize them within the peptide or protein chain. While dehydroalanine can be generated in solution, it can also be generated in the gas phase upon CID of various species. Oxidized S‐alkyl cysteine residues generate dehydroalanine upon activation via highly efficient loss of the alkyl sulfenic acid. Asymmetric cleavage of disulfide bonds upon collisional activation of systems with limited proton mobility also generates dehydroalanine. Furthermore, we show that gas‐phase ion/ion reactions can be used to facilitate the generation of dehydroalanine residues via, for example, oxidation of S‐alkyl cysteine residues and conversion of multiply‐protonated peptides to radical cations. In the latter case, loss of radical side‐chains to generate dehydroalanine from some amino acids gives rise to the possibility for residue‐specific backbone cleavage of polypeptide ions. Copyright © 2016 John Wiley & Sons, Ltd.     

Gas-phase anionic complexes of alkali metal ions and peptides: Structure and collision activated decompositions

Alkali metal ions and anionic peptides can be desorbed into the gas phase to give metal-bound peptides and bis(peptide) complexes bearing a ? 1 charge. Although amide nitrogens of peptide bonds are deprotonated in the gas phase by alkali metal ions, this reacion does not occur in solution. Metal-bound dipeptide anions exist as a single structure, whereas those of tripeptide complexes have three structures as revealed by tandem mass spectrometric studies. Ions of bis(peptide) complexes of alkali metals decompose upon collisional activation principally to form deprotonated peptides, in contrast to bis(peptide) complexes of alkaline earth metal ions, which undergo elimination of a neutral peptide.     

Synthesis of α-fluoroalkyl substituted peptides via enzymatic fragment condensation

A peptide library consisting of di- and tripeptide esters and tripeptide amides, respectively, containing α-Tfm amino acids in different positions was synthesized and tested for enzymatic fragment condensations catalyzed by the proteases α-chymotrypsin, trypsin and clostripain.     

Chemical ionization mass spectrometry of complex molecules—VI: Peptides

The chemical ionization mass spectra of a series of simple peptides containing six or fewer amino acids have been studied. Using methane as the reactant gas we found cleavage of the peptide bond occurs in two ways, yielding either the acyl carbonium ion or the complementary ammonium ion. The observation of both types of fragments permits the determination of the amino acid sequence of the peptide. The ammonium ions provide an additional sequence determining route compared to that available from electron-impact spectra. ‘Sequence-determing ions,’ especially the quasimolecular ion at m/e [M+1] are usually more intense than in the electron-impact mass spectra.     

Syntheses of thyrotropin-releasing hormones (TRH) (Schaf) and related peptides

The syntheses of seven tripeptide isomers containing L -histidine, L -proline and L -glutamic acid residues, the same as found in the natural thyrotropin-releasing hormone (TRH), are reported. In addition L -pyroglutamyl-L -histidyl-L -proline and its amide as well as N^α-acetyl-L -glutamyl-L -histidyl-L -proline are described. Whereas eight peptides are inactive and L -pyroglutamyl-L -histidyl-L -proline shows a slight TRH activity, L -pyroglutamyl-L -histidyl-L -proline-amide has the full biological activity of the isolated thyrotropin-releasing hormone and, at the present state of knowledge, seems to be identical with it.     

Gas chromatographic mass spectrometric detection of dihydroxy fatty acids preserved in the 'bound' phase of organic residues of archaeological pottery vessels

A methodology is presented for the determination of dihydroxy fatty acids preserved in the 'bound' phase of organic residues preserved in archaeological potsherds. The method comprises saponification, esterification, silica gel column chromatographic fractionation, and analysis by gas chromatography/mass spectrometry. The electron ionisation mass spectra of the trimethylsilyl ether methyl ester derivatives are characterised by fragment ions arising from cleavage of the bond between the two vicinal trimethylsiloxy groups. Other significant fragment ions are [M-15](+.), [M-31](+.), m/z 147 and ions characteristic of vicinal disubstituted (trimethylsiloxy) TMSO- groups (Δ(7,8), Δ(9,10), Δ(11,12) and Δ(13,14): m/z 304, 332, 360 and 388, respectively). The dihydroxy fatty acids identified in archaeological extracts exhibited carbon numbers ranging from C(16) to C(22) and concentrations varying from 0.05 to 14.05 μg g(-1) . The wide range of dihydroxy fatty acids observed indicates that this approach may be applied confidently in screening archaeological potsherds for the degradation products of monounsaturated fatty acids derived from commodities processed in archaeological pottery vessels.     

Characterizing Peptide Neutral Losses Induced by Negative Electron-Transfer Dissociation (NETD)

We implemented negative electron-transfer dissociation (NETD) on a hybrid ion trap/Orbitrap mass spectrometer to conduct ion/ion reactions using peptide anions and radical reagent cations. In addition to sequence-informative ladders of a•- and x-type fragment ions, NETD generated intense neutral loss peaks corresponding to the entire or partial side-chain cleavage from amino acids constituting a given peptide. Thus, a critical step towards the characterization of this recently introduced fragmentation technique is a systematic study of synthetic peptides to identify common neutral losses and preferential fragmentation pathways. Examining 46 synthetic peptides with high mass accuracy and high resolution analysis permitted facile determination of the chemical composition of each neutral loss. We identified 19 unique neutral losses from 14 amino acids and three modified amino acids, and assessed the specificity and sensitivity of each neutral loss using a database of 1542 confidently identified peptides generated from NETD shotgun experiments employing high-pH separations and negative electrospray ionization. As residue-specific neutral losses indicate the presence of certain amino acids, we determined that many neutral losses have potential diagnostic utility. We envision this catalogue of neutral losses being incorporated into database search algorithms to improve peptide identification specificity and to further advance characterization of the acidic proteome.     

Preparation of Pseudo‐Peptide Building Blocks with retro‐Thioamide Bond Mediated via Thiocarbamoyl Meldrum's Acid

An easy and efficient synthesis of pseudo‐tripeptide containing a thiomalonamide moiety was developed. Isothiocyanate derivatives of amino acids react smoothly with 2,2‐dimethyl‐1,3‐dioxane‐4,6‐dione (Meldrum's acid) to yield new thiocarbamoyl derivatives of Meldrum's acids. Thermal decomposition of these new derivatives leads to thiocarbamoyl ketenes, which acylate amino acid esters to give pseudo‐tripeptides.     

Factors affecting immonium ion intensities in the high-energy collision-induced decomposition spectra of peptides

Each amino acid in a peptide has a characteristic immonium ion (H₂N⁺?CHR), the presence of which in a mass spectrum can indicate the presence of that amino acid. High-energy collision-induced decomposition studies on small peptide ions formed by fast atom bombardment showed the relative intensities of these immonium ions to be dependent on the relative positions of the amino acids in the peptide chain: C-terminal, N-terminal or in-chain. Evidence in favour of competition in the formation of immonium ions is presented.     

A mass spectrometric and molecular orbital study of H2O loss from protonated tryptophan and oxidized tryptophan derivatives

Protonated N-acetyltryptophan, oxindolylalanine (a mono-oxidized derivative of tryptophan), and N-acetyloxindolylalanine, as well as several di- and tripeptide derivatives containing oxindolylalanine, undergo a range of fragmentation reactions in the gas phase, including the loss of water. In order to elucidate the sites of water loss within these ions, and to determine the mechanisms associated with these processes, we have conducted a series of experiments employing multistage tandem mass spectrometry (MS/MS and MS(3)) in a quadrupole ion trap mass spectrometer, regiospecific structural labeling, and independent solution-phase syntheses of proposed product ion structures, coupled with the use of molecular orbital calculations at the B3LYP/6-31G* level of theory. We demonstrate that the loss of H(2)O from the amide carbonyl group of protonated N-acetyltryptophan O-methyl ester occurs via a "side-chain-backbone" neighboring group reaction to yield a protonated carboline derivative. In contrast, the loss of water from the O-methyl ester of protonated oxindolylalanine results in the formation of a tricyclic structure by "backbone-side-chain" nucleophilic attack from the amino nitrogen to the C2 position of the indole ring. The O-methyl ester of protonated N-acetyloxindolylalanine was found to dissociate via the loss of water from both possible sites, i.e. from the side-chain indolyl oxygen and the backbone amide carbonyl group. An estimate of the relative preference for water loss from each site was obtained from the abundances of product ions formed from MS(3) analysis of regiospecifically labeled derivatives of N-acetyloxindolylalanine, and from the results of molecular orbital calculations. These studies indicate the absence of a characteristic 'signature' ion or neutral loss for peptides containing oxindolylalanine residues under low-energy ion trap CID conditions.     

Characteristic fragmentations of peptide derivatives containing proline in negative-ion fast atom bombardment mass spectrometry

Fragmentations of N-benzyloxycarbonyl-protected tripeptide ethyl esters containing proline were compared with those of the corresponding peptide derivatives not containing proline in negative-ion fast atom bombardment mass spectrometry. The fragment ion [M – 109]^? due to loss of the benzyloxy group followed by dehydrogenation from the peptide molecule was the base peak in the negative-ion mass spectra for the peptides not containing proline, whilst it was a very weak fragment ion or not observed at all in those for the peptides containing proline. These results suggest that the fragmentations of the peptide derivatives in negative-ion fast atom bombardment mass spectrometry depend on the conformational difference of the peptide derivatives owing to the existence of proline in the derivative.     

Products of Cu(II)-catalyzed oxidation of alpha-synuclein fragments containing M1-D2 and H50 residues in the presence of hydrogen peroxide

Metal-catalyzed oxidation (MCO) of proteins is mainly a site-specific process in which one or a few amino acids at metal-binding sites on the protein are preferentially oxidized. The oxidation of proteins by MCO can lead to oxidation of amino acid residue side chains, cleavage of the peptide bonds and formation of covalent protein-protein cross-linked derivatives. In an attempt to elucidate the products of the copper(II)-catalyzed oxidation of the 29-56, M29-D30-56 and Ac-M29-D30-56 fragments of alpha-synuclein, high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) methods and Cu(II)/hydrogen peroxide as a model oxidizing system were employed. The peptide solution (0.50 mM) was incubated at 37 degrees C for 24 h with metal : peptide : hydrogen peroxide 1 : 1 : 4 molar ratio in phosphate buffer, pH 7.4. Oxidation targets for all studied peptides are the histidine residues coordinated to the metal ions. For the M29-D30-56 and Ac-M29-D30-56 peptides the oxidation of the methionine residue to methionine sulfoxide and sulfone is observed. The cleavage of the peptide bond M29-D30 for the M29-D30-56 peptide was detected as metal binding residues. The fragmentations of the M29-D30-56 peptide near the Lys residues were observed supporting the participation of this (Lys) residue in the coordination of the copper(II) ions.     

Collision-induced dissociation of alkali metal cationized and permethylated oligosaccharides: Influence of the collision energy and of the collision gas for the assignment of linkage position

Tandem mass spectrometry has been used to study the collision-induced decomposition of [M+Na]⁺ ions of permethylated oligosaccharides. It is shown that many linkage positions in one compound may be determined by the presence or absence, in a single spectrum, of specific fragment ions that arise from the cleavage of two ring bonds and that the yield of such ions depends strongly on the collision energy and nature of the collision gas. In contrast to the behavior of monolithiated native oligosaccharides, the collision-induced decomposition of the sodiated and permethylated oligosaccharide samples at low energy leads to preferential cleavage of glycosidic linkages. At high collision energies, the fragment ions formed by cleavage of more than one bond are greatly enhanced, especially when helium is replaced by argon as the collision gas. Furthermore, argon is the more efficient collision gas in inducing fragmentation of the precursor ions. As an example of the application of this method, the discrimination between the 1 → 3 and 1 → 6-linked mannose residues in the common core of N-glycans is described.