Novel natural peptides from Hyla arborea schelkownikowi skin secretion

Hyla arborea schelkownikowi is one of the leaf frog species inhabiting the southern territories of Russia and the former USSR. This frog species is a member of the Hylidae Rafinesque, 1815 batrachians family. The present study deals with the previously uninvestigated peptidome of the Hyla arborea schelkownikowi skin secretion. Nano‐electrospray ionization Fourier transform mass spectrometry (nanoESI‐FTMS) of the skin secretion, in the intact form and after acetylation, was selected as the general method of analysis. Electron‐capture dissociation (ECD) and collision‐induced dissociation (CID) fragmentation were both employed, while de novo sequencing was performed by manual interpretation of the MS data. The suppression of the cyclization of b‐ions in the mass spectrometer by the acetylation reaction proved to be very efficient for the de novo sequencing of short peptides. Ten skin peptides were found and all of them, except for bradykinin, had not previously been reported. Six of the peptides belong to the tryptophyllins and related peptides, while three peptides are similar to the aureins. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of a peptide family from the skin secretion of the Middle East Tree Frog Hyla savignyi by composition‐based de novo sequencing

A new tryptophyllin‐like peptide family was found in the skin secretion of the tree frog Hyla savignyi. Peptides were characterized by database‐independent sequencing strategies and specific ion fragmentation features were investigated. Skin secretions from specimens of Hyla savignyi were collected by mild electrical stimulation. Peptides were separated by reversed‐phase nano‐high‐performance liquid chromatography (nanoHPLC) and mass spectra were acquired online by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR‐MS). Peptides were characterized by manual de novo sequencing and by composition‐based sequencing (CBS), appearing mostly as C‐terminal free acids and as their acid amide analogs. Amide peptides yielded lower intensities of y‐type ions after collision‐induced dissociation (CID) than their acid analogs. A mechanism of internal b‐ion formation (positive ion mode) and of CO₂ elimination (negative ion mode) is proposed. We also exemplified phenomena such as the proline effect and formation of non‐direct sequence ions after sequence rearrangements. The occurrence of rearrangement products, of internal ions and of the proline effect made the CID spectra highly complex. CBS analysis nevertheless resulted in successful and highly reliable sequence analysis. Copyright © 2010 John Wiley & Sons, Ltd.     

Isolation and sequence analysis of peptides from the skin secretion of the Middle East tree frog Hyla savignyi

Novel peptides were identified in the skin secretion of the tree frog Hyla savignyi. Skin secretions were collected by mild electrical stimulation. Peptides were separated by reversed-phase high-performance liquid chromatography. Mass spectra were acquired by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), and fragment ion spectra were obtained after collision-induced dissociation and electron capture dissociation. Peptides were analyzed by manual de novo sequencing and composition-based sequencing (CBS). Sequence analyses of three so far undescribed, structurally unrelated peptides are presented in this paper, having the sequences DDSEEEEVE-OH, P*EEVEEERJK-OH, and GJJDPJTGJVGGJJ-NH₂. The glutamate-rich sequences are assumed to be acidic spacer peptides of the prepropeptide. One of these peptides contains the modified amino acid hydroxyproline, as identified and localized by high-accuracy FTICR-MS. Combination of CBS and of experience-based manual sequence analysis as complementary and database-independent sequencing strategies resulted in peptide identification with high reliability.

De novo peptide sequencing by tandem MS using complementary CID and electron transfer dissociation

De novo sequencing of peptides using tandem MS is difficult due to missing fragment ions in the spectra commonly obtained after CID of peptide precursor ions. Complementing CID spectra with spectra obtained in an ion‐trap mass spectrometer upon electron transfer dissociation (ETD) significantly increases the sequence coverage with diagnostic ions. In the de novo sequencing algorithm CompNovo presented here, a divide‐and‐conquer approach was combined with an efficient mass decomposition algorithm to exploit the complementary information contained in CID and ETD spectra. After optimizing the parameters for the algorithm on a well‐defined training data set obtained for peptides from nine known proteins, the CompNovo algorithm was applied to the de novo sequencing of peptides derived from a whole protein extract of Sorangium cellulosum bacteria. To 2406 pairs of CID and ETD spectra contained in this data set, 675 fully correct sequences were assigned, which represent a success rate of 28.1%. It is shown that the CompNovo algorithm yields significantly improved sequencing accuracy as compared with published approaches using only CID spectra or combined CID and ETD spectra.     

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.     

Cyclization of peptide b9 ions

The product ion mass spectra obtained by CID of the b₉ ions derived by loss of neutral alanine from the MH⁺ ion of the peptides Tyr(Ala)₉, (Ala)₄Tyr(Ala)₅, and (Ala)₈TyrAla are essentially identical, indicative of full cyclization reaction to a common intermediate before fragmentation. This leads to abundant nondirect sequence ions in the product ion mass spectra of the b₉ ions. The product ion mass spectra of the b₈ ions from the first two peptides also are essentially identical. The fragmentation of the MH⁺ ions also leads to low intensity nondirect sequence ions in the product ion mass spectra. N-terminal acetylation blocks the cyclization and eliminates nondirect sequence fragment ions in the product ion mass spectra.     

Characterization of N‐methylated amino acids by GC‐MS after ethyl chloroformate derivatization

Methylation is an essential metabolic process in the biological systems, and it is significant for several biological reactions in living organisms. Methylated compounds are known to be involved in most of the bodily functions, and some of them serve as biomarkers. Theoretically, all α‐amino acids can be methylated, and it is possible to encounter them in most animal/plant samples. But the analytical data, especially the mass spectral data, are available only for a few of the methylated amino acids. Thus, it is essential to generate mass spectral data and to develop mass spectrometry methods for the identification of all possible methylated amino acids for future metabolomic studies. In this study, all N‐methyl and N,N‐dimethyl amino acids were synthesized by the methylation of α‐amino acids and characterized by a GC‐MS method. The methylated amino acids were derivatized with ethyl chloroformate and analyzed by GC‐MS under EI and methane/CI conditions. The EI mass spectra of ethyl chloroformate derivatives of N‐methyl ( 1–18 ) and N,N‐dimethyl amino acids ( 19–35 ) showed abundant [M‐COOC₂H₅]⁺ ions. The fragment ions due to loss of C₂H₄, CO₂, (CO₂ + C₂H₄) from [M‐COOC₂H₅]⁺ were of structure indicative for 1–18 . The EI spectra of 19–35 showed less number of fragment ions when compared with those of 1–18 . The side chain group (R) caused specific fragment ions characteristic to its structure. The methane/CI spectra of the studied compounds showed [M + H]⁺ ions to substantiate their molecular weights. The detected EI fragment ions were characteristic of the structure that made easy identification of the studied compounds, including isomeric/isobaric compounds. Fragmentation patterns of the studied compounds ( 1–35 ) were confirmed by high‐resolution mass spectra data and further substantiated by the data obtained from ¹³C₂‐labeled glycines and N‐ethoxycarbonyl methoxy esters. The method was applied to human plasma samples for the identification of amino acids and methylated amino acids. Copyright © 2016 John Wiley & Sons, Ltd.     

Utility of reaction intermediate monitoring with photodissociation multi-stage (MS) time-of-flight mass spectrometry for mechanistic and structural studies: Phosphopeptides

In tandem mass spectra of phosphopeptides, intact sequence ions are often missing or appear weakly. Instead, dephosphorylated sequence ions appear prominently. In this work, we used photodissociation (PD) multi-stage (MSⁿ) time-of-flight mass spectrometry that can monitor reaction intermediates with lifetime as short as 100 ns to study the formation of dephosphorylated sequence ions such as y_n-H₃PO₄. y_n-H₃PO₄ was found to be formed mainly by H₃PO₄ loss from y_n. For doubly phosphorylated peptides, y_n seemed to lose H₃PO₄ stepwise and form y_n-H₃PO₄ and y_n-2H₃PO₄. Even when y_n was absent in PD-MS² spectrum, its m/z could be predicted from those of y_n-H₃PO₄ and/or y_n-2H₃PO₄. Complete sequence coverage was possible when the data from PD-MS² and PD-MS³ were combined, demonstrating the utility of transient ion detection by PD-MS³ for structure analysis.     

Controlling the structure of sequence‐defined poly(phosphodiester)s for optimal MS/MS reading of digital information

Digital polymers are monodisperse chains with a controlled sequence of co‐monomers, defined as letters of an alphabet, and are used to store information at the molecular level. Reading such messages is hence a sequencing task that can be efficiently achieved by tandem mass spectrometry. To improve their readability, structure of sequence‐controlled synthetic polymers can be optimized, based on considerations regarding their fragmentation behavior. This strategy is described here for poly(phosphodiester)s, which were synthesized as monodisperse chains with more than 100 units but exhibited extremely complex dissociation spectra. In these polymers, two repeating units that differ by a simple H/CH₃ variation were defined as the 0 and 1 bit of the ASCII code and spaced by a phosphate moiety. They were readily ionized in negative ion mode electrospray but dissociated via cleavage at all phosphate bonds upon collisional activation. Although allowing a complete sequence coverage of digital poly(phosphodiester)s, this fragmentation behavior was not efficient for macromolecules with more than 50 co‐monomers, and data interpretation was very tedious. The structure of these polymers was then modified by introducing alkoxyamine linkages at appropriate location throughout the chain. A first design consisted of placing these low dissociation energy bonds between each monomeric bit: while cleavage of this sole bond greatly simplified MS/MS spectra, efficient sequencing was limited to chains with up to about 50 units. In contrast, introduction of alkoxyamine bonds between each byte (i.e. a set of eight co‐monomers) was a more successful strategy. Long messages (so far, up to 8 bytes) could be read in MS³ experiments, where single‐byte containing fragments released during the first activation stage were further dissociated for sequencing. The whole sequence of such byte‐truncated poly(phosphodiester)s could be easily re‐constructed based on a mass tagging system which permits to determine the original location of each byte in the chain. Copyright © 2017 John Wiley & Sons, Ltd.     

The computer-assisted carboxypeptidase method for C-terminal sequencing of proteins and polypeptides

On further study of the computer-assisted carboxypeptidase method for sequencing the C-terminals of proteins or peptides, two computer programs—DPS and CPA were successfully developed on VAX-11/780 computer and tested with some synthetic peptides and the degradation fragment of the natural protein as model substrates. The C-terminal sequence of CB-3, one of the CNBr degradation fragments of trichosanthin, had first been determined by this method as: -Ser-Ala-Ser-Ala-Ala-Leu-Hse-OH, which was later confirmed by other ways of sequencing. This method is not only able to extend the C-terminal sequencing up to 7 amino acid residues, but also useful for determining the C-terminal sequence with repeating amino acid residues.     

Stapled Peptides with γ‐Methylated Hydrocarbon Chains for the Estrogen Receptor/Coactivator Interaction

“Stapled” peptides are typically designed to replace two non‐interacting residues with a constraining, olefinic staple. To mimic interacting leucine and isoleucine residues, we have created new amino acids that incorporate a methyl group in the γ‐position of the stapling amino acid S5. We have incorporated them into a sequence derived from steroid receptor coactivator 2, which interacts with estrogen receptor α. The best peptide (IC₅₀=89 nm ) replaces isoleucine 689 with an S‐γ‐methyl stapled amino acid, and has significantly higher affinity than unsubstituted peptides (390 and 760 nm ). Through X‐ray crystallography and molecular dynamics studies, we show that the conformation taken up by the S‐γ‐methyl peptide minimizes the syn‐pentane interactions between the α‐ and γ‐methyl groups.     

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.     

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.     

A Systematic Study of Acidic Peptides for b-Type Sequence Scrambling

A systematic study was carried out to examine the effects of acidic amino acid residues and the position of the acidic group on the cyclization of b ions. The study utilized the model C-terminal amidated peptides XAAAAAA, AXAAAAA, AAXAAAA, AAAXAAA, AAAAXAA, AAAAAXA, AAAAAAX, XXAAAAAA, AAXXAAAA, AAAAXXAA, and AAAAAAXX, where X is a glutamic acid (E) or aspartic acid (D) residue. The CID mass spectra of b _n (where n = 7 and 8) ions derived from XAAAAAA, AAAXAAA, AAAAAAX and XXAAAAAA, AAXXAAAA, AAAAXXAA, and AAAAAAXX exhibited very similar fragmentation patterns for both the glutamic and the aspartic acid peptide series. The CID mass spectra of MH⁺ derived from model peptides presented substantial direct and non-direct sequence b ions. The results indicate that b ions produced from acidic peptides can also undergo head-to-tail cyclization, which is the reason for the formation of the non-direct sequence b ions. The b ion spectra derived from the peptides became more complex as the number of acidic residues in the peptides increased. Side chains of glutamic and aspartic acid did not inhibit the cyclization of the b ions. Substantial water elimination was observed in all CID spectra of b ₇ and b ₈ ions. Finally, the preferential cleavage of glutamic or aspartic acid residues from macrocyclic structures of b ions was also investigated under various collision energy conditions.     

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.     

Mass spectrometric study of peptides secreted by the skin glands of the brown frog Rana arvalis from the Moscow region

A high‐performance liquid chromatography nano‐electrospray ionization Fourier transform mass spectrometry (HPLC/nanoESI‐FTMS) approach involving recording of collision‐activated dissociation (CAD) and electron‐capture dissociation (ECD) spectra of an intact sample and two its modifications after performic oxidation and reduction followed by carboxamidomethylation helps to establish peptide profiles in the crude secretion of frog species at mid‐throughput level, including de novo sequencing. The proposed derivatization procedures allow increasing of the general sequence coverage in the backbone, providing complementary information and, what is more important, reveal the amino acid sequence in the cystine ring (‘rana box’). Thus purely mass spectrometric efficient sequencing becomes possible for longer than usual proteolytic peptides. Seventeen peptides belonging to four known families were identified in the secretion of the European brown frog Rana arvalis inhabiting the Moscow region in Russia. Ranatuerins, considered previously a unique feature of the North American species, as well as a new melittin‐related peptide, are worth special mention. The developed approach was previously successfully used for the identification of peptides in the skin secretion of the Caucasian green frog Rana ridibunda. Copyright © 2009 John Wiley & Sons, Ltd.     

On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis

Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300–1800. Good focusing was achieved for insulin receptor, which produced ∼10 s peak width. For 0.1 mg mL⁻¹ bovine serum albumin (BSA) digests, 24 ± 1 peptides (sequence coverage 47 ± 4%) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts.     

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.     

De novo sequence analysis and intact mass measurements for characterization of phycocyanin subunit isoforms from the blue‐green alga Aphanizomenon flos‐aquae

In this work, partial characterization of the primary structure of phycocyanin from the cyanobacterium Aphanizomenon flos‐aquae (AFA) was achieved by mass spectrometry de novo sequencing with the aid of chemical derivatization. Combining N‐terminal sulfonation of tryptic peptides by 4‐sulfophenyl isothiocyanate (SPITC) and MALDI‐TOF/TOF analyses, facilitated the acquisition of sequence information for AFA phycocyanin subunits. In fact, SPITC‐derivatized peptides underwent facile fragmentation, predominantly resulting in y‐series ions in the MS/MS spectra and often exhibiting uninterrupted sequences of 20 or more amino acid residues. This strategy allowed us to carry out peptide fragment fingerprinting and de novo sequencing of several peptides belonging to both α‐ and β‐phycocyanin polypeptides, obtaining a sequence coverage of 67% and 75%, respectively. The presence of different isoforms of phycocyanin subunits was also revealed; subsequently Intact Mass Measurements (IMMs) by both MALDI‐ and ESI‐MS supported the detection of these protein isoforms. Finally, we discuss the evolutionary importance of phycocyanin isoforms in cyanobacteria, suggesting the possible use of the phycocyanin operon for a correct taxonomic identity of this species. Copyright © 2008 John Wiley & Sons, Ltd.     

Sequencing of peptide‐derived Amadori products by the electron capture dissociation method

The electron capture dissociation (ECD) of peptide‐derived Amadori products has been successfully applied for their sequencing. In contrast to the collision induced dissociation (CID), based on the vibrational excitation of peptides, the ECD method does not produce ions formed by fragmentation of the hexose moiety, that facilitates interpretation of the obtained spectra. The fragmentation spectrum is dominated by c_n and z·_n ions, providing the sufficient information for sequencing of peptides and establishing the location of glycated Lys residues in the peptide chain. The ECD experiments were conducted on a series of synthetic peptides and unseparated digests of glycated ubiquitin. Copyright © 2009 John Wiley & Sons, Ltd.