Amino acid identification and sequence analysis of peptides by reaction mass spectrometry

Fast atom bombardment mass spectrometry (FAB-MS) is applied to distinguish N-terminal series ions from C-terminal series ions of a peptide by on-probe acetylation, it providesvaluable information about the sequence of an unknown peptide. The FAB mass spectra containa number of characteristic ions at low-mass region in addition to the sequence ions at high-massregion. It was found that the ions below m/z 200 are characteristic of the amino acid composition ofthe peptide, from which the amino acid composition of the peptide could be estimated. Additionally,mixture analysis is also discussed.     

ESI‐MS/MS analysis of protonated N‐methyl amino acids and their immonium ions

Methylation is one of the important posttranslational modifications of biological systems. At the metabolite level, the methylation process is expected to convert bioactive compounds such as amino acids, fatty acids, lipids, sugars, and other organic acids into their methylated forms. A few of the methylated amino acids are identified and have been proved as potential biomarkers for several metabolic disorders by using mass spectrometry–based metabolomics workstation. As it is possible to encounter all the N‐methyl forms of the proteinogenic amino acids in plant/biological systems, it is essential to have analytical data of all N‐methyl amino acids for their detection and identification. In earlier studies, we have reported the ESI‐MS/MS data of all methylated proteinogenic amino acids, except that of mono‐N‐methyl amino acids. In this study, the N‐methyl amino acids of all the amino acids ( 1 ‐ 21 ; including one isomeric pair) were synthesized and characterized by ESI‐MS/MS, LC/MS/MS, and HRMS. These data could be useful for detection and identification of N‐methyl amino acids in biological systems for future metabolomics studies. The MS/MS spectra of [M + H]⁺ ions of most N‐methyl amino acids showed respective immonium ions by the loss of (H₂O, CO). The other most common product ions detected were [MH‐(NH₂CH₃]⁺, [MH‐(RH)]⁺ (where R = side chain group) ions, and the selective structure indicative product ions due to side chain and N‐methyl group. The isomeric/isobaric N‐methyl amino acids could easily be differentiated by their distinct MS/MS spectra. Further, the MS/MS of immonium ions inferred side chain structure and methyl group on α‐nitrogen of the N‐methyl amino acids.     

Low-mass ions produced from peptides by high-energy collision-induced dissociation in tandem mass spectrometry

High-energy collision-induced dissociation (CID) mass spectrometry provides a rapid and sensitive means for determining the primary sequence of peptides. The low-mass region (below mass 300) of a large number of tandem CID spectra of peptides has been analyzed. This mass region contains several types of informative fragment ions, including dipeptide ions, immonium ions, and other related ions. Useful low-mass ions are also present in negative-ion CID spectra. Immonium ions (general structure [H₂N=CH-R]⁺, where R is the amino acid side chain) and related ions characteristic of specific amino acid residues give information as to the presence or absence of these residues in the peptide being analyzed. Tables of observed immonium and reiated ions for the 20 standard amino acids and for a number of modified amino acids are presented. A database consisting of 228 high-energy CID spectra of peptides has been established, and the frequency of occurrence of various ions indicative of specific ammo acid residues has been determined. Two model computer-aided schemes for analysis of the ammo-acid content of unknown peptides have been developed and tested against the database.     

A linear free-energy correlation in the low-energy tandem mass spectra of protonated tripeptides Gly–Gly–Xxx

The backbone cleavages of protonated tripeptide ions of the series Gly—Gly—Xxx, where Xxx ? Gly, Ala, Val, d-Leu, l-Leu, Ile, Phe, Tyr, Trp, Pro, Met and Glu, were studied in a hybrid tandem mass spectrometer. C-Terminal y-type ions and N-terminal a- and b-type ions were noted. A linear relationship between log (y₁/b₂) and the proton affinity of the C-terminal amino acid substituents was found: as the proton affinity of the C-terminal residue increases, the fraction of y₁ ion formation increases. When the C-terminal substituent was more basic than Trp, the b₂ ion was not observed. It is likely that the site of protonation changes from peptide bond to side-chain for just these residues, Lys, His and Arg.     

Tandem mass spectrometry of peptides: Mechanistic aspects and structural information based on neutral losses. II—Tri- and larger peptides

The neutral products arising during the collisionally activated dissociation of protonated oligopeptides (MH⁺) are post-ionized by collision and detected in neutral fragment-reionization (⁺N_fR⁺) mass spectra. For the isomeric tripeptides Ala-Gly-Gly, Gly-Ala-Gly and Gly-Gly-Ala, the amino acid and dipeptide losses from the C-terminus and the diketopiperazine losses from the N-terminus allow for differentiation. These neutral fragments are identified in the corresponding ⁺N_fR⁺ spectra by comparison to reference collision-induced dissociative ionization (CIDI) mass spectra of individual amino acids, dipeptides and diketopiperazines. Peptides with distinct C-termini but otherwise identical sequences are found to yield ⁺N_fR⁺ products that are characteristic of the respective C-terminal amino acid. This is demonstrated for several peptide pairs, including leucine- and methionine-enkephalin. In general, ⁺N_fR⁺ spectra are dominated by the heavier neutral losses; further, ⁺N_fR⁺ and CIDI cause extensive dissociation, indicating that the collisional ionization process imparts high average internal energies.     

A comparison of the peptide fragmentation obtained from a reflector matrix-assisted laser desorption-ionization time-of-flight and a tandem four sector mass spectrometer

The types, extent, and overall distribution of peptide fragmentation produced by matrix-assisted laser desorption-ionization-postsource decay (MALDI-PSD) on a reflector time-of-flight mass spectrometer were compared with those obtained from high and low energy collision-induced dissociation (CID) on a four-sector mass spectrometer and from liquid secondary ion mass spectrometry (LSIMS) ion source fragmentation and LSIMS metastable ion (MI) decomposition on a two-sector mass spectrometer. The model peptides studied had sequences and compositions that yielded predominantly either N- or C-terminal fragmentation from CID. For des-Arg¹ and des-Arg⁹ bradykinin (i.e., H-PPGFSPFR-OH and H-RP-PGFSPF-OH, respectively), the types of fragment ions and the extent to which each type is formed in both MALDI-PSD and low energy CID spectra are remarkably similar. This observation suggests that both methods deposit comparable internal energies (IE) into [M + H]⁺ precursor ions. The distribution of N-terminal, C-terminal, immonium, and internal fragmentation from MALDI-PSD spectra of des-Arg¹ and des-Arg⁹ bradykinin did not change dramatically with respect to the terminal arginine position, contrary to those from LSIMS MI decomposition, high and low energy CID spectra. This observation in combination with the prominent immonium, internal, and minus 17 fragment ion types in PSD indicates that the imparted IE from MALDI and the 14 µs of flight time may promote steady-state decomposition kinetics. Fragmentation distributions of MALDI-PSD spectra are also similar to those in LSIMS spectra. This implies that the distribution of protonation sites in [M + H]⁺ is comparable for both techniques.     

Amino Acid Sequence of N-Terminal Peptide of Normal Human Serum Albumin

From the peptic digest of normal human serum albumin., the N-terminal peptide comprising 24 amino acid residues was obtained by means of peptide mapping. Combined uses of trypsin, α-chymotrypsin, thermolysin, carboxypeptidase A and Dansyl-Edman technique resulted in the elucidation of amino acid sequence of no. 1 to no. 24 as follows: NH₂-Asp-Ala-His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys-Asp-Leu-GIy-Glu-Glu-Asn-Phe-Lys-Ala-Leu-Val-Leu-COOH These sequence results agree completely with those recently published by other workers.     

Polymeric hydrogels modified with ornithine and lysine: Sorption and release of metal cations and amino acids

A novel, convenient synthesis, using copper ions, is described for the multigram‐scale preparation of acryloyl and methacryloyl ornithine and lysine without the need to use protecting groups and chromatographic purifications. Three methods of removing the copper ions from the amino acid derivatives were examined. The obtained acryloyl and methacryloyl ornithine and lysine were copolymerized with N‐isopropylacrylamide and N,N′‐methylenebisacrylamide as crosslinking agents, resulting in a series of hydrogels with varying incorporated amino acid content. The relative content of a given amino acid was estimated from the ¹H NMR data and compared with its molar fraction used in the polymerization process. We investigated the influence of the amount of amino acid groups incorporated into the polymer network on the swelling behavior of the gels in the presence of metal ions of different ability to form complexes (Cu²⁺, Co²⁺, and Ca²⁺) with α‐amino acid groups and the sorption of copper ions. Next, the presence of α‐amino acid groups attached to the polymer network was used to bond the compounds which can cocomplex metal ions. Phenylalanine was selected for examination of its cocomplexation of Cu²⁺ with the polymer‐network amino acids and its consecutive release from the gel after appropriate change of pH. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Sequencing of cyclosporins by fast atom bombardment and linked-scan mass spectrometry

The mass spectrometric sequence determination of amino acid residues in cyclosporins using fast atom bombardment, collisionally activated dissociations in the first field-free region and linked B/E scan is described. The general fragmentation scheme was derived from the spectra of cyclosporins A, B, C, D, F, G, L and [DH-MeBmt¹]CS. The main fragmentation pathways start by primary splitting between amino acids 2–3, 1–11 and 5–6. The corresponding N-terminal b-type ions are common fragment types in the mass spectra. The 1–11 splitting can be enhanced by the introduction of a lactone group into the peptide ring by conversion of cyclosporins into isocyclosporins. The fragmentation scheme was used for amino acid sequence determination in four new natural cyclosporins, [MeLeu¹]CS, [Leu⁴]CS, [Ile⁴]CS and [Leu⁵]CS.     

Studies on enzymic peptide synthesis: I. Synthesis of tryptophane analogue of Leu-enkephalin

A tryptophane analogue of Leu-enkephalin, Tyr-Gly-Gly-Trp-Leu-NH₂, was synthesized by stepwise elongation from C-terminal Leucylamide towards the N-terminal tyrosine using α-chymotrypsin and papain catalysis. The selections of appropriate enzymes and suitable solvents were studied. It was found that an N-blocked amino acid ester served better as a donor than the corresponding free amino acid in the peptide formation, irrespective of whether α-chymotrypsin or papain was used, while, on the other hand, an N-blocked amino acid was needed as the donor in case of thermolysin. In the last step of pentapeptide synthesis catalyzed by α-chymotrypsin, the phase transfer process seems to be preferable to the homogeneous solution method. The results of this work showed some advantages by the use of an immobilized enzyme. The tryptophane analogue of Leu-enkephalin was also synthesized by the conventional organic method. Preliminary in vitro bioassay indicated that the synthetic Try-Gly-Gly-Try-Leu-NH₂ can inhibit the contraction of the guinea Pig's ileum caused by electric stimulation to nearly the same extent as the natural Leu-enkephalin does.     

An algorithm for estimating the enthalpies of formation of amine molecular ions and immonium fragment ions

A method of estimating the enthalpies of formation of amino molecular ions, ΔH^? ([C_nH_2n+1NH₂]⁺˙) and of immonium ions ΔH([C_nH_2n+1N]⁺) is reported. It is based on the fact that CH₃ is isoelectronic with NH₂, CH₂ with NH and CH with N. Some calculated values of the enthalpies of formation of amine molecular ions and immonium ions are reported and estimates are made of the accuracy of such calculation.     

In situ N-phosphorylation of oligopeptides for fast atom bombardment mass spectrometry

Positive ion fast atom bombardment mass spectrometry (FABMS) of in situ N-phosphorylated oligopeptides showed intense quasi-molecular ions together with the successive alkene loss fragment ions, which afford multiple checks of the unequivocal reality of the relative molecular mass of the tested samples. More interesting, in a novel cleavage pattern only the N-phosphoryl fragment ions gave intense peaks, the C-terminal series ions being suppressed. For each of the N-terminal ions, losses of alkenes also occur to provide multiple checks for the existence of these ions. The FABMS of the in situ N- phosphorylated oligopeptides might provide an easily accessible routine method for peptide sequencing.     

Mapping <Emphasis Type="Italic">N</Emphasis>-terminus phosphorylation sites and quantitation by stable isotope dimethyl labeling

We have previously coupled stable isotope dimethyl labeling with IMAC enrichment for quantifying the extent of protein phosphorylation in vivo. The enhanced a₁ signal of dimethylated peptides served as a unique mass tag for unequivocal identification of the N-terminal amino acids. In this study, we demonstrate that the a₁ ion could further assist in mapping the precise phosphorylation site near the N-terminal region and allow the determination of the exact site and level of phosphorylation in one step by stable isotope dimethyl labeling. We show that the a₁ ion signal was suppressed for dimethylated peptides with a phosphorylation site at the N-terminus Ser/Thr residue (N-p*Ser/Thr) but was still enhanced for N-terminus Tyr residue (N-p*Tyr) or internal Ser/Thr residues (-p*Ser/Thr). Based on the dominant de-phosphorylated molecular ions and b-H₃PO₄ ions for N-p*Ser/Thr, we propose that dimethyl labeling increases the basicity of the N-terminus and accelerates the de-phosphorylation for N-p*Ser/Thr precursors, which, however, suppresses the a₁ ion enhancement due to the resulting unsaturated covalent bond on C _α of the N-terminus amino acid. Using this method, we excluded three Ser/Thr phosphorylation sites in A431 cells, two of which, however, were previously reported to be phosphorylation sites; we confirmed three known phosphorylation sites in A431 cells and quantified their ratios upon EGF treatment. Notably, we identified a novel phosphorylation site on Ser43 residue at N-terminus of the tryptic peptide derived from SVH protein in pregnant rat uteri. SVH protein has not been reported or implied with any phosphorylation event, and our data show that the Ser43 of SVH is an intrinsic phosphorylation site in pregnant rat uteri and that its phosphorylation level was slightly decreased upon c-AMP treatment.     

Characterization of N,N‐dimethyl amino acids by electrospray ionization–tandem mass spectrometry

Methylation is an essential metabolic process for a number of critical reactions in the body. Methyl groups are involved in the healthy function of the body life processes, by conducting methylation process involving specific enzymes. In these processes, various amino acids are methylated, and the occurrence of methylated amino acids in nature is diverse. Nowadays, mass‐spectrometric‐based identification of small molecules as biomarkers for diseases is a growing research. Although all dimethyl amino acids are metabolically important molecules, mass spectral data are available only for a few of them in the literature. In this study, we report synthesis and characterization of all dimethyl amino acids, by electrospray ionization–tandem mass spectrometry (MS/MS) experiments on protonated molecules. The MS/MS spectra of all the studied dimethyl amino acids showed preliminary loss of H₂O + CO to form corresponding immonium ions. The other product ions in the spectra are highly characteristic of the methyl groups on the nitrogen and side chain of the amino acids. The amino acids, which are isomeric and isobaric with the studied dimethyl amino acids, gave distinctive MS/MS spectra. The study also included MS/MS analysis of immonium ions of dimethyl amino acids that provide information on side chain structure, and it is further tested to determine the N‐terminal amino acid of the peptides. Copyright © 2015 John Wiley & Sons, Ltd.     

Charge States of y Ions in the Collision-Induced Dissociation of Doubly Charged Tryptic Peptide Ions

Bonds that break in collision-induced dissociation (CID) are often weakened by a nearby proton, which can, in principle, be carried away by either of the product fragments. Since peptide backbone dissociation is commonly charge-directed, relative intensities of charge states of product y- and b-ions depend on the final location of that proton. This study examines y-ion charge distributions for dissociation of doubly charged peptide ions, using a large reference library of peptide ion fragmentation generated from ion-trap CID of peptide ions from tryptic digests. Trends in relative intensities of y²⁺ and y¹⁺ ions are examined as a function of bond cleavage position, peptide length (n), residues on either side of the bond and effects of residues remote from the bond. It is found that y_n-2/b₂ dissociation is the most sensitive to adjacent amino acids, that y²⁺/y¹⁺ steadily increase with increasing peptide length, that the N-terminal amino acid can have a major influence in all dissociations, and in some cases other residues remote from the bond cleavage exert significant effects. Good correlation is found between the values of y²⁺/y¹⁺ for the peptide and the proton affinities of the amino acids present at the dissociating peptide bond. A few deviations from this correlation are rationalized by specific effects of the amino acid residues. These correlations can be used to estimate trends in y²⁺/y¹⁺ ratios for peptide ions from amino acid proton affinities.     

Effect of ligand on the redox reactions of thallium metal clusters

Pulse radiolysis of an aqueous solution of mono-valent thallium ion and mixed solutions of Tl⁺/Ag⁺ in the presence of various amino polycarboxylic acids such as trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTPA), N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA) and triethylenetetraminehexaacetic acid (TTHA) has been carried out. Abnormal valence states of Tl ions were generated. It is concluded that DCTA, DTPA, HEDTA and TTHA decrease the redox potential of Tl ions in aqueous solutions. It was observed that the electron transfer from complexed Tl₂⁺ to Ag⁺ varied in the range 7.5 × 10⁸ to 1.0 × 10⁹, depending on the type of complexing ligand. Electron transfer from Tl₂⁺ to Ag⁺ lead to the formation of silver atoms, which agglomerate further to form silver colloid.     

Factors Affecting the Production of Aromatic Immonium Ions in MALDI 157 nm Photodissociation Studies

Immonium ions are commonly observed in the high energy fragmentation of peptide ions. In a MALDI-TOF/TOF mass spectrometer, singly charged peptides photofragmented with 157 nm VUV light yield a copious abundance of immonium ions, especially those from aromatic residues. However, their intensities may vary from one peptide to another. In this work, the effect of varying amino acid position, peptide length, and peptide composition on immonium ion yield is investigated. Internal immonium ions are found to have the strongest intensity, whereas immonium ions arising from C-terminal residues are the weakest. Peptide length and competition among residues also strongly influence the immonium ion production. Quantum calculations provide insights about immonium ion structures and the fragment ion conformations that promote or inhibit immonium ion formation.

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Graphical Abstract ?

     

Der 2-Trimethylsilyläthyl-Rest als selektiv abspaltbare Carboxy-Schutzgruppe

The 2-trimethylsilylethyl residue, a selectively cleavable carboxyl protecting group In a search for new carboxyl protecting groups suitable for use in peptide synthesis, 2-trimethylsilylethyl esters [-COOCH₂CH₂Si(CH₃)₃] of several N-protected amino acids have been prepared. These esters can be synthesized in good yields from N^a-benzyloxycarbonyl-amino acids and 2-trimethylsilylethanol with dicyclohexylcarbodiimide in the presence of pyridine. They are stable under a wide variety of conditions used during coupling and work-up in peptide synthesis. For removal the 2-trimethylsilylethyl group is readily cleaved by fluoride ions, preverably using a quaternary ammonium fluoride in dimethylformamide. Some side reactions which occurred during the removal of the 2-trimethylsilylethyl group are discussed. Special attention has been paid to the question of racemization during the treatment with fluoride ions. No. evidence of racemization was found in any of the cases examined.     

Shifting unoccupied spectral space in mass spectrum of peptide fragment ions

Ions near the high-end border of a mass defect distribution plot for native peptide fragment ions have potential as signature markers that are based on mass-to-charge ratio determination. The specificity of these marker ions, including phosphoryl ions, can be improved by removing interfering isobaric ions from the border region on the distribution plot. These interfering ions are rich in Asp and Glu content. The masses of amino acid residues and peptides are rescaled from the IUPAC scale (¹²C=12 u as the mass reference) to the averagine scale (averagine mass=111 u* as the mass reference with zero mass defect; u*: the mass unit on the averagine scale), using a scaling factor of 0.999493894. It is theoretically predicted that esterification of Asp and Glu side-chain carboxylates with n-butanol can achieve a sufficient retreat of the high-end border on a mass defect distribution plot based on the use of mass spectrometers with better-than-medium resolution. Theoretical calculations and laboratory experiments are performed to examine effects of various esterifications on the averagine-scale mass defect distribution of peptide fragment ions and on the specificity of two positive phosphoryl ions: the phosphotyrosine immonium ion and a cyclophosphoramidate ion.     

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.