Analysis of cyclic pyrolysis products formed from amino acid monomer

Amino acid was mixed with silica and tetramethylammonium hydroxide (TMAH) to favor pyrolysis of amino acid monomer. The pyrolysis products formed from amino acid monomer were using GC/MS and GC. 20 amino acids of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine were analyzed. The pyrolysis products were divided into cyclic and non-cyclic products. Among the 20 amino acids, arginine, asparagine, glutamic acid, glutamine, histidine, lysine, and phenylalanine generated cyclic pyrolysis products of the monomer. New cyclic pyrolysis products were formed by isolation of amino acid monomers. They commonly had polar side functional groups to 5-, 6-, or 7-membered ring structure. Arginine, asparagine, glutamic acid, glutamine, histidine, and phenylalanine generated only 5- or 6-membered ring products. However, lysine generated both 6- and 7-membered ring compounds. Variations of the relative intensities of the cyclic pyrolysis products with the pyrolysis temperature and amino acid concentration were also investigated.     

Characterization of the dehydration products due to thermal decomposition of peptides by liquid chromatography‐tandem mass spectrometry

Thermal decomposition (TD) of proteins is being investigated as a rapid digestion step for bottom‐up proteomics. Mass spectrometry (MS) analyses of the TD products of simple peptides and intact proteins have revealed several nonvolatile products at masses lower than the precursor biomolecule (M). In addition to products stemming from site‐specific cleavages, many signals are also observed at a corresponding M‐18, most likely because of dehydration (M‐H₂O) during the heating process. Understanding the structural nature of the water loss product is important in establishing the utility of their tandem mass spectra (collision‐induced dissociation) in determining the precursor ion amino acid sequence in a bottom‐up proteomic workflow. Dehydration of a peptide can take place from a variety of sources including side chain groups, C‐terminus, and/or intramolecular cyclization (C to N‐terminus cyclization). In this work, liquid chromatography‐tandem MS (LC‐MS/MS) and a series of standard peptides (angiotensin II, DRVYIHPF and its cyclic analog) are implemented to decipher the structure of the TD dehydration product. In addition, a derivatization strategy incorporating N‐terminus acetylation was developed that allowed the direct comparison of tandem mass spectra of standard cyclic peptides with those resulting from the TD process, thus eliminating any ambiguity from the direct comparison of their mass spectra (due to gas‐phase cyclization of b‐ions, which can result in sequence scrambling of the precursor ion). Results from these investigations indicated that peptide dehydrated TD products were mostly linear in nature, and water loss was favored from the C‐terminus carboxyl group or, when present, the aspartic acid side chain. Given the predictable nature of the formation of TD dehydration products, their MS/MS analysis can be of utility in providing complementary and confirmatory sequence information of the precursor peptide. Copyright © 2015 John Wiley & Sons, Ltd.     

Primary and secondary locations of charge sites in angiotensin II (M + 2H)2+ ions formed by electrospray ionization

High-energy tandem mass spectrometry and molecular dynamics calculations are used to determine the locations of charge in metastably decomposing (M + 2H)2+ ions of human angiotensin II. Charge-separation reactions provide critical information regarding charge sites in multiple charged ions. The most probable kinetic energy released (Tm.p.) from these decompositions are obtained using kinetic energy release distributions (KERDs) in conjunction with MS/MS (MS2), MS/MS/MS (MS3), and MS/MS/MS/MS (MS4) experiments. The most abundant singly and doubly charged product ions arise from precursor ion structures in which one proton is located on the arginine (Arg) side chain and the other proton is located on a distal peptide backbone carbonyl oxygen. The MS3 KERD experiments show unequivocally that neither the N-terminal amine nor the aspartic acid (Asp) side chain are sites of protonation. In the gas phase, protonation of the less basic peptide backbone instead of the more proximal and basic histidine (His) side chain is favored as a result of reduced coulomb repulsion between the two charge sites. The singly and doubly charged product ions of lesser abundance arise from precursor ion structures in which one proton is located on the Arg side chain and the other on the His side chain. This is demonstrated in the MS3 and MS4 mass-analyzed ion kinetic energy spectrometry experiments. Interestingly, (b7" + OH)2+ product ions, like the (M + 2H)2+ ions of angiotensin II, are observed to have at least two different decomposing structures in which charge sites have a primary and secondary location.     

Analytical pyrolysis of dipeptides containing proline and amino acids with polar side chains. Novel 2,5-diketopiperazine markers in the pyrolysates of proteins

The formation of cyclic dipeptides (DKPs, 2,5-diketopiperazines) from dipeptides having proline (Pro) as the fixed N-terminal amino acid was investigated by analytical pyrolysis with a heated platinum filament coil. Glutamic acid (Glu), aspartic acid (Asp), glutamine (Gln), lysine (Lys) or arginine (Arg) was the terminal amino acid. Products evolved from off-line pyrolysis at 500 °C were analysed by GC–MS as such or after trimethylsilylation. The structure of a novel DKP from the pyrolysis of Pro-Glu, namely hexahydrodipyrrolo[1,2-a:1′,2′-d]pyrazine-3,5,10(10aH)-trione, was established by ESI-MS/MS and extensive NMR analysis. This DKP could be identified in the pyrolysates of dipeptide Pro-Gln, tripeptide Pro-Glu-Leu, collagen and bovine serum albumin (BSA). Pyrolysis of Pro-Lys and Lys-Pro-Leu afforded the cyclo(Pro-Lys) tentatively identified by interpretation of its trimethylsilyl (TMS) derivative mass spectra and the DKPs resulting from the deamination of the lateral chain. The dipeptide Pro-Asp yielded isomeric cyclo(Pro-Asp) revealed as TMS derivatives and cyclo(Pro-Ala) from the decarboxylation of the side chain. Analytical pyrolysis of the above peptides as well as of Pro-Tyr, collagen and BSA enabled the compilation of GC (retention times relative to an internal standard) and MS data (characteristic ions) of over eighty DKPs including their TMS derivatives, the latter ones useful in the identification of thermal degradation products of proteinaceous materials in several matrices.     

Malondialdehyde tagging improves the analysis of arginine oligomers and arginine-containing dendrimers by HPLC-MS

The selective modification of arginine residues by malondialdehyde (MDA) was used to improve the mass spectrometric analysis of arginine oligomers (Arg(x), x = 4, 6, 7, 8, 9) and an arginine-containing dendrimeric peptide. MDA tagging significantly increased the hydrophobicity of the arginine side-chain and resulted in improved retention in RP HPLC of the oligoarginines using formic acid as mobile phase additive. This avoided the use of TFA to generate sufficient retention, as TFA was shown to lead to a dramatically reduced sensitivity (up to ten-fold for Arg(8) and Arg(9)) as a result of the strong signal suppression by ion pairing with multiple basic residues. MDA modification of Arg oligomers not only resulted in improved detection sensitivity for most of the peptides studied (e. g., more than six-fold for Arg(7)), but also greatly enhanced the quality of MS/MS spectra, in line with previous results for other peptides. Furthermore, MDA modification helped to identify major side products in a sample of a dendrimeric peptide, a class of peptides that is typically difficult to analyze by MS.     

Microwave-assisted reaction of peptide formation by amino acid with phosphate:Exploration of the most possible channels for the origin of life

The formation of peptides and then protein by small abiological molecules clusters such as amino acid is a key stage in the origin of life[1]. More and more ex- perimental results showed that phosphate plays an important role in the formation of biomolecules in prebiotic chemical evolution. The principal reservoirs of biochemical energy are phosphates (such as ATP). The peculiar role of phosphates in contemporary life might suggest its essential role in prebiotic energy conversion, syntheses …     

Independent highly sensitive characterization of asparagine deamidation and aspartic acid isomerization by sheathless CZE‐ESI‐MS/MS

Amino acids residues are commonly submitted to various physicochemical modifications occurring at physiological pH and temperature. Post‐translational modifications (PTMs) require comprehensive characterization because of their major influence on protein structure and involvement in numerous in vivo process or signaling. Mass spectrometry (MS) has gradually become an analytical tool of choice to characterize PTMs; however, some modifications are still challenging because of sample faint modification levels or difficulty to separate an intact peptide from modified counterparts before their transfer to the ionization source. Here, we report the implementation of capillary zone electrophoresis coupled to electrospray ionization tandem mass spectrometry (CZE‐ESI‐MS/MS) by the intermediate of a sheathless interfacing for independent and highly sensitive characterization of asparagine deamidation (deaN) and aspartic acid isomerization (isoD). CZE selectivity regarding deaN and isoD was studied extensively using different sets of synthetic peptides based on actual tryptic peptides. Results demonstrated CZE ability to separate the unmodified peptide from modified homologous exhibiting deaN, isoD or both independently with a resolution systematically superior to 1.29. Developed CZE‐ESI‐MS/MS method was applied for the characterization of monoclonal antibodies and complex protein mixture. Conserved CZE selectivity could be demonstrated even for complex samples, and foremost results obtained showed that CZE selectivity is similar regardless of the composition of the peptide. Separation of modified peptides prior to the MS analysis allowed to characterize and estimate modification levels of the sample independently for deaN and isoD even for peptides affected by both modifications and, as a consequence, enables to distinguish the formation of l ‐aspartic acid or d ‐aspartic acid generated from deaN. Separation based on peptide modification allowed, as supported by the ESI efficiency provided by CZE‐ESI‐MS/MS properties, and enabled to characterize and estimate studied PTMs with an unprecedented sensitivity and proved the relevance of implementing an electrophoretic driven separation for MS‐based peptide analysis. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization and Diagnostic Value of Amino Acid Side Chain Neutral Losses Following Electron-Transfer Dissociation

Using a large set of high mass accuracy and resolution ETD tandem mass spectra, we characterized ETD-induced neutral losses. From these data we deduced the chemical formula for 20 of these losses. Many of them have been previously observed in electron-capture dissociation (ECD) spectra, such as losses of the side chains of arginine, aspartic acid, glutamic acid, glutamine, asparagine, leucine, histidine, and carbamidomethylated cysteine residues. With this information, we examined the diagnostic value of these amino acid-specific losses. Among 1285 peptide–spectrum matches, 92.5% have agreement between neutral loss-derived peptide amino acid composition and the peptide sequences. Moreover, we show that peptides can be uniquely identified by using only the accurate precursor mass and amino acid composition based on neutral losses; the median number of sequence candidates from an accurate mass query is reduced from 21 to 8 by adding side chain loss information. Besides increasing confidence in peptide identification, our findings suggest the potential use of these diagnostic losses in ETD spectra to improve false discovery rate estimation and to enhance the performance of scoring functions in database search algorithms.     

Negative ion production from peptides and proteins by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Negative ion production from peptides and proteins was investigated by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Although most research on peptide and protein identification with ionization by MALDI has involved the detection of positive ions, for some acidic peptides protonated molecules are not easily formed because the side chains of acidic residues are more likely to lose a proton and form a deprotonated species. After investigating more than 30 peptides and proteins in both positive and negative ion modes, [M–H]⁻ ions were detected in the negative ion mode for all peptides and proteins although the matrix used was 2,5‐dihydroxybenzoic acid (DHB), which is a good proton donor and favors the positive ion mode production of [M+H]⁺ ions. Even for highly basic peptides without an acidic site, such as myosin kinase inhibiting peptide and substance P, good negative ion signals were observed. Conversely, gastrin I (1‐14), a peptide without a highly basic site, will form positive ions. In addition, spectra obtained in the negative ion mode are usually cleaner due to absence of alkali metal adducts. This can be useful during precursor ion isolation for MS/MS studies. Copyright © 2008 John Wiley & Sons, Ltd.     

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

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

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.     

The ornithine effect in peptide cation dissociation

Facile cleavage C‐terminal to ornithine residues in gas phase peptides has been observed and termed the ornithine effect. Peptides containing internal or C‐terminal ornithine residues, which are formed from deguanidination of arginine in solution, were fragmented to produce either a y‐ion or water loss, respectively, and the complementary b‐ion. The fragmentation patterns of several peptides containing arginine were compared to those of the ornithine analogues. Conversion of arginine to ornithine results in a decrease of the gas phase proton affinity of the residue, thereby increasing the mobility of the ionizing proton. This alteration allows the nucleophilic amine to facilitate a neighboring group reaction to induce a cleavage of the adjacent amide bond. The selective cleavage at the ornithine residue is proposed to result from the highly favorable generation of a six‐membered lactam ring. The ornithine effect was compared with the well‐known proline and aspartic acid effects in peptide fragmentation using angiotensin II, DRVYIHPF and the ornithine analogue, DOVYIHPF. Under conditions favorable to either the aspartic acid (i.e. singly protonated peptide) or proline effect (i.e. doubly protonated peptide), the ornithine effect was consistently observed to be the more favorable fragmentation pathway. The highly selective nature of the ornithine effect opens up the possibility for conversion of arginine to ornithine residues to induce selective cleavages in polypeptide ions. Such an approach may complement strategies that seek to generate non‐selective cleavages of the related peptides. Copyright © 2013 John Wiley & Sons, Ltd.     

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

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

Improving fragmentation of poorly fragmenting peptides and phosphopeptides during collision-induced dissociation by malondialdehyde modification of arginine residues

Despite significant technological and methodological advancements in peptide sequencing by mass spectrometry, analyzing peptides that exhibit only poor fragmentation upon collision-induced dissociation (CID) remains a challenge. A major cause for unfavorable fragmentation is insufficient proton 'mobility' due to charge localization at strongly basic sites, in particular, the guanidine group of arginine. We have recently demonstrated that the conversion of the guanidine group of the arginine side chain by malondialdehyde (MDA) is a convenient tool to reduce the basicity of arginine residues and can have beneficial effects for peptide fragmentation. In the present work, we have focused on peptides that typically yield incomplete sequence information in CID-MS/MS experiments. Energy-resolved tandem MS experiments were carried out on angiotensins and arginine-containing phosphopeptides to study in detail the influence of the modification step on the fragmentation process. MDA modification dramatically improved the fragmentation behavior of peptides that exhibited only one or two dominant cleavages in their unmodified form. Neutral loss of phosphoric acid from phosphopeptides carrying phosphoserine and threonine residues was significantly reduced in favor of a higher abundance of fragment ions. Complementary experiments were carried out on three different instrumental platforms (triple-quadrupole, 3D ion trap, quadrupole-linear ion trap hybrid) to ascertain that the observation is a general effect.     

Helical peptide models for protein glycation: proximity effects in catalysis of the Amadori rearrangement.

INTRODUCTION: Non-enzymatic glycation of proteins has been implicated in various diabetic complications and age-related disorders. Proteins undergo glycation at the N-terminus or at the epsilon-amino group of lysine residues. The observation that only a fraction of all lysine residues undergo glycation indicates the role of the immediate chemical environment in the glycation reaction. Here we have constructed helical peptide models, which juxtapose lysine with potentially catalytic residues in order to probe their roles in the individual steps of the glycation reaction. RESULTS: The peptides investigated in this study are constrained to adopt helical conformations allowing residues in the i and i+4 positions to come into spatial proximity, while residues i and i+2 are far apart. The placing of aspartic acid and histidine residues at interacting positions with lysine modulates the steps involved in early peptide glycation (reversible Schiff base formation and its subsequent irreversible conversion to a ketoamine product, the Amadori rearrangement). Proximal positioning of aspartic acid or histidine with respect to the reactive lysine residue retards initial Schiff base formation. On the contrary, aspartic acid promotes catalysis of the Amadori rearrangement. Presence of the strongly basic residue arginine proximate to lysine favorably affects the pK(a) of both the lysine epsilon-amino group and the singly glycated lysine, aiding in the formation of doubly glycated species. The Amadori product also formed carboxymethyl lysine, an advanced glycation endproduct (AGE), in a time-dependent manner. CONCLUSIONS: Stereochemically defined peptide scaffolds are convenient tools for studying near neighbor effects on the reactivity of functional amino acid sidechains. The present study utilizes stereochemically defined peptide helices to effectively demonstrate that aspartic acid is an efficient catalytic residue in the Amadori arrangement. The results emphasize the structural determinants of Schiff base and Amadori product formation in the final accumulation of glycated peptides.     

Role of the site of protonation in the low-energy decompositions of gas-phase peptide ions

The dissociation of singly or multiply protonated peptide ions by using low-energy collisional activation (CA) is highly dependent on the sites of protonation. The presence of strongly basic amino acid residues in the peptide primary structure dictates the sites of protonation, which generates a precursor ion population that is largely homogeneous with respect to charge sites. Attempts to dissociate this type of precursor ion population by low-energy CA result in poor fragmentation via few pathways. The work described here represents a systematic investigation of the effects of charge heterogeneity in the precursor ion population of a series of model peptides in low-energy CA experiments. Incorporation of acidic residues in the peptide RLC*IFSC*FR (where C* indicates a cysteic acid residue), for example, balances the charge on the basic arginine residues, which enables the ionizing protons to reside on a number of less basic sites along the peptide backbone. This results in a precursor ion population that is heterogeneous with respect to charge site. Low-energy CA of these ions results in diverse and efficient fragmentation. Molecular modeling has been utilized to demonstrate that energetically preferred conformations incorporate an intraionic interaction between arginine and cysteic acid residues.     

Characteristics of photodissociation at 193 nm of singly protonated peptides generated by matrix-assisted laser desorption ionization (MALDI)

Photodissociation (PD) at 193 nm of various singly protonated peptides was investigated. These include peptides with an arginine residue at the C-terminus, N-terminus, at both termini, inside the chain, and those without an arginine residue. Monoisotopomeric selection was made for the precursor ions. Interference from the post-source decay (PSD) product signals was reduced as much as possible by using the deflection system (reported previously) and subtracting the remaining signals from the laser-on signals. The presence of an arginine residue and its position inside the peptide were found to significantly affect the PD spectra, as reported previously. Presence of a proline, aspartic acid, or glutamic acid residue hardly affected the PD spectral patterns. By comparing the PD spectra obtained at a few different wavelengths, it is concluded that the dissociation of the photoexcited ions occurs in their ground electronic states. Tentative explanations for the observed spectral correlations based on the statistical picture for the reactions are also presented.     

Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (b_n-1 + H₂O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H₂O and NH₃) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment ion in the high mass range of the MS/MS spectra. The mass difference between this signal and the protonated molecular ion corresponds to the mass of the C-terminal residue. It allowed a straightforward identification of the amino acid positioned at this extremity. It must be emphasized that a neutral residue loss can be misattributed to the formation of a y_m-1 ion, i.e., to the loss of the N-terminal residue following the a₁-y_m–1 fragmentation channel. Extreme caution must be adopted when reading the direct sequence ion on the positive ion MS/MS spectra of singly charged peptides not to mix up the attribution of the N- and C-terminal amino acids. Although such peculiar fragmentation behavior is of obvious interest for de novo peptide sequencing, it can also be exploited in proteomics, especially for studies involving digestion protocols carried out with proteolytic enzymes other than trypsin (Lys-N, Glu-C, and Asp-N) that produce arginine-containing peptides.     

Quantitative analysis of complex amino acids and RGD peptides by X‐ray photoelectron spectroscopy (XPS)

The C 1 s, N 1 s, and O 1 s core level binding energies (BEs) of the functional groups in amino acids (glycine, aspartic acid, glutamic acid, arginine, and histidine) with varied side‐chains and cell‐binding RGD‐based peptides have been determined and characterized by X‐ray photoelectron spectroscopy with a monochromatic Al K_α source. The zwitterionic nature of the amino acids in the solid state is unequivocally evident from the N 1 s signals of the protonated amine groups and the C 1 s signature of carboxylate groups. Significant adventitious carbon contamination is evident for all samples but can be quantitatively accounted for. No intrinsic differences in the XP spectra are evident between two polymorphs (α and γ) of glycine, indicating that the crystallographic differences have a minor influence on the core level BEs for this system. The two nitrogen centers in the imidazole group of histidine exhibit an N 1 s BE shift that is in line with previously reported data for theophylline and aqueous imidazole solutions, while the nitrogen and carbon chemical shifts reflect the unusual guanidinium chemical environment in arginine. It is shown that the complex envelopes of C 1 s and O 1 s photoemission spectra for short‐chain peptides can be analyzed quantitatively by reference to the less complex XP spectra of the constituent amino acids, provided the peptides are of high enough purity. The distinctive N 1 s photoemission from the amide linkages provides an indicator of peptide formation even in the presence of common impurities, and variations in the relative intensities of N 1 s were found to be diagnostic for each of the three peptides investigated (RGD, RGDS, and RGDSC). Copyright © 2013 John Wiley & Sons, Ltd.     

Differentiating alpha- and beta-aspartic acids by electrospray ionization and low-energy tandem mass spectrometry

Spectra obtained by low-energy electrospray ionization tandem mass spectrometry (ESI-MS/MS) of 34 peptides containing aspartic acids at position n were studied and unambiguously differentiated. beta-Aspartic acid yields an internal rearrangement similar to that of the C-terminal rearrangements of protonated and cationized peptides. As a result of this rearrangement, two different ions containing the N- and the C-terminal ends of the original peptide are formed, namely, the bn-1 + H2O and y"l - n + 1 - 46 ions, respectively, where e is the number of amino acid residues in the peptide. The structure suggested for the y"l - n + 1 - 46 ion is identical to that proposed for the vn ions observed upon high-energy collision-induced dissociation (CID) experiments. The intensity of these ions in the low-energy MS/MS spectra is greatly influenced by the presence and position of basic amino acids within the sequences. Peptides with a basic amino acid residue at position n - 1 with respect to the beta-aspartic acid yield very intense bn-1 + H2O ions, while the y"l - n + 1 - 46 ion was observed mostly in tryptic peptides. Comparison between the high- and low-energy MS/MS spectra of several isopeptides suggests that a metastable fragmentation process is the main contributor to this rearrangement, whereas for long peptides (40 AA) CID plays a more important role. We also found that alpha-aspartic acid containing peptides yield the normal immonium ion at 88 Da, while peptides containing beta-aspartic acid yield an ion at m/z 70, and a mechanism to explain this phenomenon is proposed. Derivatizing isopeptides to form quaternary amines, and performing MS/MS on the sodium adducts of isopeptides, both improve the relative intensity of the bn + 1 + H2O ions. Based on the above findings, it was possible to determine the isomerization sites of two aged recombinant growth proteins.