Electrospray ionization tandem mass spectrometry sequencing of novel skin peptides from Ranid frogs containing disulfide bridges

Tandem mass spectrometry sequencing, as well as Edman sequencing of peptides belonging to the Rana genus, represents a difficult task due to the presence of a disulfide bridge at the C-terminus and their rather high molecular masses (over 2000 Da). The present study throws light upon the sequence of three rather long peptides (more than 20 amino acid residues each) isolated from the skin secretion of Russian frogs, Rana ridibunda and Rana arvalis. This novel aspect involves the fact that the sequences (including two sequences established de novo) were determined exclusively by means of mass spectrometry. A combination of electron capture dissociation (ECD) and collision-induced dissociaiton (CID) data accompanied by exact mass measurements (LTQ Fourier transform ion cyclotron resonance mass spectrometer) facilitated reaching the goal. To overcome the difficulty dealing with disulphide bridges ("Rana box"), reduction of the S-S bond with dithiotreitol followed by derivatization of Cys residues with iodoacetamide was used. The sequence was determined using combined spectral data on y and b series of fragment ions. A multiple mass spectrometry (MS(3)) experiment was also used to elucidate the sequence inside the "Rana box" after cysteine derivatization. Exact mass measurements were used to differentiate between Lys and Gln residues, while characteristic losses of 29 and 43 Da (d and w fragment ions) in CID and ECD experiments allowed us to distinguish between Ile and Leu isomeric acids.     

A tris (2-carboxyethyl) phosphine (TCEP) related cleavage on cysteine-containing proteins

Introduced in the late 1980s as a reducing reagent, Tris (2-carboxyethyl) phosphine (TCEP) has now become one of the most widely used protein reductants. To date, only a few studies on its side reactions have been published. We report the observation of a side reaction that cleaves protein backbones under mild conditions by fracturing the cysteine residues, thus generating heterogeneous peptides containing different moieties from the fractured cysteine. The peptide products were analyzed by high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptides with a primary amine and a carboxylic acid as termini were observed, and others were found to contain amidated or formamidated carboxy termini, or formylated or glyoxylic amino termini. Formamidation of the carboxy terminus and the formation of glyoxylic amino terminus were unexpected reactions since both involve breaking of carbon—carbon bonds in cysteine.     

Characterisation of the peptaibiome of the biocontrol fungus Trichoderma atroviride by liquid chromatography/tandem mass spectrometry

The present study describes the liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based screening and characterisation of linear antibiotic alpha-aminoisobutyric acid (Aib)-containing non-ribosomal peptides (NRP) in culture samples of the filamentous fungus Trichoderma atroviride ATCC 74058. Fungal culture filtrates were enriched by solid-phase extraction (SPE) and separated by reversed-phase high-performance liquid chromatography (HPLC), prior to mass spectrometric (MS) and tandem mass spectrometric (MS/MS) analysis on a triple quadrupole-linear ion trap tandem mass spectrometer. A workflow consisting of two alternative screening strategies was applied to search for NRP. Various MS full scan and MS/MS measurement modes led to the identification of 16 trichorzianines and diagnostic in-source fragment ions of another four trichorzianines. Furthermore, we detected 15 novel Aib-containing peptides with putative molecular weights ranging from 951.7 to 1043.7 g/mol (monoisotopic masses), composed of up to 9 amino acids. While the amino acid sequences of the novel peptaibiotics showed typical microheterogeneity and consisted of the amino acids Leu/Ile, Aib, Ser, Val/Iva, Gly, Ac-Aib, Tyr and Phe, the mass increments at the C-termini of the peptides were not assignable to any residues described in the literature. The amino acid sequences were confirmed and structure proposals made for both molecule termini by high-resolution MS and MS/MS analysis. We propose the group name 'trichoatrokontins' for the newly identified peptaibiotics. As no other peptaibiotics were found in the culture samples, the peptaibiome of the investigated strain of T. atroviride consists of at least 20 trichorzianines and 15 trichoatrokontins.     

Identification of Photooxidation Sites in Bovine α-Crystallin

Abstract— Because UV irradiation of proteins can produce reactive oxygen species and exposure to UV light has been implicated in cataractogenesis, the sites of photooxidation of bovine α-crystallin, a major lens protein with molecular chaperone activity, were identified using tandem mass spectrometry (MS/MS). Bovine α-crystallin was irradiated with UV light (293 nm) for 1, 4 and 8 h, digested with trypsin and analyzed by matrix-assisted laser de-sorption ionization, time-of-flight mass spectrometry (MALDI) to identify the oxidized sequences. Tryptic peptides were purified by reverse-phase HPLC and oxidized peptides were sequenced by MS/MS to determine the sites of oxidation. Tryptophan fluorescence decreased exponentially with increasing time of UV exposure and peptides containing residues 1-11 of α-crystallin and 1-11, 12-22 and 57-69 of α-crystallin were determined to be oxidized by shifts of 16 D or multiples of 16 Da above the mass of the unmodified peptide. The MALDI analysis revealed single oxidation of all four sequences, which increased with increasing time of UV exposure and possible double oxidation of α 12-22. The specific sites of photooxidation indicate that the N-terminal regions of α-and β-crystallin are exposed to an aqueous environment and are in the vicinity of tryptophan residues from neighboring subunits.     

Effect of pH on the interfacial adsorption activity of pulmonary surfactant

The effect of pH on the interfacial adsorption activity of pulmonary surfactant was examined. Measurements of the surface tension were made in a Wilhelmy-like surface microbalance specially designed to assay small volumes of hypophase in thermostatically controlled conditions. Alkaline pH caused a significant decrease of the surface activity of both pulmonary surfactant and a lipid extract from surfactant (LES) (containing all of the lipids and surfactant protein-B (SP-B) and surfactant protein-C (SP-C) hydrophobic surfactant proteins, but lacking surfactant protein-A). The pK calculated from the change of surface activity versus pH was 9.18±0.26 and 9.27±0.31 for pulmonary surfactant and LES, respectively. The results from this study support the idea that electrostatic interactions between basic residues of SP-B and SP-C and negatively charged surfactant phospholipids could be important for the interfacial adsorption activity of pulmonary surfactant.     

Screening for disulfide-rich peptides in biological sources by carboxyamidomethylation in combination with differential matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Peptides with biological functions often contain disulfide bridges connecting two cysteine residues. In an attempt to screen biological fluids for peptides containing cysteine residues, we have developed a sensitive and specific method to label cysteines selectively and detect the resulting molecular mass shift by differential mass spectrometry. First, reduction of disulfide bridges and carboxyamidomethylation of free thiols is adjusted to quantitatively achieve cysteine alkylation for complex peptide extracts. In a second step, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) before and after chemical derivatization is performed, followed by differential analysis to determine shifted peaks; shifted peaks belong to cysteine-containing peptides, other peaks remain unchanged. The number of cysteines can then be determined by the resulting molecular mass shift. Free, reduced cysteines are shifted by 57 u, two oxidized cysteines involved in disulfide bridges (cystine) result in a shift to higher mass per disulfide bridge of 116 u. Disulfide bridges connecting different amino acid chains like insulin break up during reduction. In this case, two peaks with lower molecular masses result from a single one in the unmodified sample. With this technique, we were able to identify cysteine-containing peptides and short fragments of proteins present in human blood filtrate.     

Tandem mass spectrometry of kahalalides: identification of two new cyclic depsipeptides, kahalalide R and S from Elysia grandifolia

Spectra obtained using electrospray ionization mass spectrometry (ESI-MS) of the mollusk Elysia grandifolia showed a cluster of molecular ion peaks centered at a molecular mass of 1478 Da (kahalalide F, an anticancer agent). Two new molecules, kahalalide R (m/z 1464) and S (m/z 1492) were characterized using tandem mass spectrometry. The mass differences of 14 Da suggest that they are homologous molecules. In addition, previously identified kahalalide D and kahalalide G are also reported. However, the ESI-MS of the mollusk's algal diet Bryopsis plumosa showed the presence of only kahalalide F. The amino acid sequences of kahalalide R and S are proposed using collision-induced dissociation (CID) experiments of singly and doubly charged molecular ions and by comparison with the amino acid sequence of kahalalide F. The pathway is presented for the loss of amino acid residues in kahalalide F. It is observed that there is sequential loss of amino acids in the linear peptide chain, but in the cyclic part the ring opens at the amide bond rather than at the lactone linkage, and the loss of amino acid residues is not sequential. The CID experiment of the alkali-metal-cationized molecular ions shows that the sodium and potassium ions coordinate to the amide nitrogen/oxygen in the linear peptide chain of the molecule and not to the lactone oxygen of the lactone. In the case of kahalalide D, CID of the protonated peptide opens the depsipeptide ring to form a linear peptide with acylium ion, and fragment ion signals indicate losses of amino acids in sequential order. In this study, tandem mass spectrometry has provided the detailed information required to fully characterize the new peptides.     

Novel phosphoryl derivatization method for peptide sequencing by electrospray ionization mass spectrometry

A one-step phosphoryl derivatization method has been used in a peptide sequencing procedure for electrospray ionization tandem mass spectrometry (ESI-MS/MS). The sodiated derivatized peptides exhibit very simple dissociation patterns, in which two kinds of fragment ions, [b(n) + OH + Na]+ and [a(n) + Na]+, are formed. Since the amino acid residues are lost sequentially from the C-terminus, peptide sequences can be identified easily. The fragmentation efficiency of peptides increased as a result of the phosphorylation, and also provided peaks of useful intensity at lower m/z. A peptide with lysine at the C-terminus was derivatized and analyzed by ESI-MS/MS. Similar mass spectra, from which the sequence could be read out, were obtained. This is a novel derivatization method yielding neutral derivatives that should be suitable for peptide sequencing by LC/ESI-MS/MS.     

Reproducible microwave-assisted acid hydrolysis of proteins using a household microwave oven and its combination with LC-ESI MS/MS for mapping protein sequences and modifications

A new set-up for microwave-assisted acid hydrolysis (MAAH) with high efficiency and reproducibility to degrade proteins into peptides for mass spectrometry analysis is described. It is based on the use of an inexpensive domestic microwave oven and can be used for low volume protein solution digestion. This set-up has been combined with liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI QTOF MS) for mapping protein sequences and characterizing phosphoproteins. It is demonstrated that for bovine serum albumin (BSA), with a molecular mass of about 67,000 Da, 1292 peptides (669 unique sequences) can be detected from a 2 μg hydrolysate generated by trifluoroacetic acid (TFA) MAAH. These peptides cover the entire protein sequence, allowing the identification of an amino acid substitution in a natural variant of BSA. It is shown that for a simple phosphoprotein containing one phosphoform, β-casein, direct analysis of the hydrolysate generates a comprehensive peptide map that can be used to identify all five known phosphorylation sites. For characterizing a complex phosphoprotein consisting of different phosphoforms with varying numbers of phosphate groups and/or phosphorylation sites, such as bovine α_s1-casein, immobilized metal-ion affinity chromatography (IMAC) is used to enrich the phosphopeptides from the hydrolysate, followed by LC-ESI MS analysis. The MS/MS data generated from the initial hydrolysate and the phosphopeptide-enriched fraction, in combination with MS analysis of the intact protein sample, allow us to reveal the presence of three different phosphoforms of bovine α_s1-casein and assign the phosphorylation sites to each phosphoform with high confidence.     

Isotope-labeled cross-linkers and fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex

For structural studies of proteins and their complexes, chemical cross-linking combined with mass spectrometry presents a promising strategy to obtain structural data of protein interfaces from low quantities of proteins within a short time. We explore the use of isotope-labeled cross-linkers in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for a more efficient identification of cross-linker containing species. For our studies, we chose the calcium-independent complex between calmodulin and a 25-amino acid peptide from the C-terminal region of adenylyl cyclase 8 containing an "IQ-like motif." Cross-linking reactions between calmodulin and the peptide were performed in the absence of calcium using the amine-reactive, isotope-labeled (d0 and d4) cross-linkers BS3 (bis[sulfosuccinimidyl]suberate) and BS2G (bis[sulfosuccinimidyl]glutarate). Tryptic in-gel digestion of excised gel bands from covalently cross-linked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. In cases where more than one reactive functional group, e.g., amine groups of lysine residues, is present in a sequence stretch, MS/MS analysis is a prerequisite for unambiguously identifying the modified residues. MS/MS experiments revealed two lysine residues in the central alpha-helix of calmodulin as well as three lysine residues both in the C-terminal and N-terminal lobes of calmodulin to be cross-linked with one single lysine residue of the adenylyl cyclase 8 peptide. Further cross-linking studies will have to be conducted to propose a structural model for the calmodulin/peptide complex, which is formed in the absence of calcium. The combination of using isotope-labeled cross-linkers, determining the accurate mass of intact cross-linked products, and verifying the amino acid sequences of cross-linked species by MS/MS presents a convenient approach that offers the perspective to obtain structural data of protein assemblies within a few days.     

栝楼蛋白 2: 栝楼蛋白部分化学结构的初步测定

栝楼蛋白(Trichobitacin)是从栝楼(Trichosanthes kirilowiiMaxim, Cucurbitaceae)中新发现的核糖体失活蛋白, 分子量为27,228; pI为9.6。应用基质辅助的激光解析飞行时间质谱(MALDI-TOF-MS)和快原子轰击质谱法(FAB-MS)分别测定胰蛋白酶酶解栝楼蛋白和天花粉蛋白(Trichosanthin)的混合肽质谱, 通过比较发现了一些分子量相同的肽。由于这两种蛋白质都来源于栝楼块根, 同源性比较强, 所以这些肽序列在两种蛋白质中基本一样; 再结合蛋白N-端自动顺序仪测定栝楼蛋白N-端的结果, 确定了栝楼蛋白N-端38个氨基酸的顺序, 栝楼蛋白经胰蛋白酶酶解后所得肽段用HPLC分离纯化, 再用蛋白质自动顺序仪, DABITC/PITC双偶合手工法和质谱法共确定了栝楼蛋白N-端, C-端等100多个氨基酸残基的序列。     

Electron detachment dissociation for top-down mass spectrometry of acidic proteins

Electron detachment dissociation (EDD) is an emerging mass spectrometry (MS) technique for the primary structure analysis of peptides, carbohydrates, and oligonucleotides. Herein, we explore the potential of EDD for sequencing of proteins of up to 147 amino acid residues by using top-down MS. Sequence coverage ranged from 72% for Melittin, which lacks carboxylic acid functionalities, to 19% for an acidic 147-residue protein, to 12% for Ferredoxin, which showed unusual backbone fragmentation next to cysteine residues. A limiting factor for protein sequencing by EDD is the facile loss of small molecules from amino acid side chains, in particular CO(2). Based on the types of fragments observed and fragmentation patterns found, we propose detailed mechanisms for protein backbone cleavage and side chain dissociation in EDD. The insights from this study should further the development of EDD for top-down MS of acidic proteins.     

Structural study of PLGA [copoly (DL-lactic/glycolic acid)], a biodegradable polymer for parenteral sustained release preparations, by tandem mass spectrometry.

A copoly (DL-lactic/glycolic acid) (PLGA), with a weight-average molecular weight of about 8400, has been characterized using fast atom bombardment (FAB)-tandem mass spectrometry in order to determine the sequence. Because of the large molecular size, PLGA was partially hydrolyzed and the terminal hydroxyl groups in the resulting oligomer mixture acetylated as the indicator. The FAB spectrum of this sample showed a complex ion signal pattern containing monomer to octamer. Diagnostic product ions containing useful information for sequence determination were observed in collision-induced dissociation-MS/MS and MS/MS/MS of these oligomer ions. The results of analysis for dimers through pentamers showed that they have random sequences of lactic and glycolic acid, suggesting that the whole structure of PLGA also has a random sequence.     

New method to study the effects of peptide sequence on the dissociation energetics of peptide ions

A new method has been developed to study the dissociation patterns of singly protonated peptides by using a quadrupole ion trap mass spectrometer. The new approach involves using boundary-activated dissociation to characterize the ease of dissociation of peptide ions. Insight can be gained into the effect of specific peptide sequences on the dissociation energetics of protonated peptides. Increased knowledge of the effects of specific sequences on the dissociation patterns of peptide ions should improve the ability to interpret complex spectra from tandem mass spectrometry (MS/MS) experiments. This method has confirmed the previously observed increase in the energy needed for the dissociation of peptide ions containing basic residues. In addition, this technique has revealed the effect of the location of proline residues on the dissociation energetics of peptides with this amino acid.     

Determination of N-linked glycosylation of yeast external invertase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The extent of N-glycosylation of yeast external invertase at each of the 14 potential sites was determined by the combination of proteolytic digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS). The average molecular mass of the intact external invertase was determined as 97 kDa by MALDI/TOF-MS. The intact protein was digested with trypsin, Lys-C and Asp-N, followed by high-performance liquid chromatographic separation. The proteolytic digests were analyzed by MALDI/MS screening for the glycopeptides. The glycopeptides were then treated with peptide:N-glycosidase F (PNGase F) and/or endo-beta-N-acetylglucosaminidase (Endo H) and the molecular mass of the deglycosylated peptide was determined by MALDI/MS and matched with the peptide predicted by a computer program. The sequences of some peptides or deglycosylated peptides were identified by the MALDI post-source decay technique. The size of the oligosaccharide, the degree of glycosylation and the distribution of the oligosaccharides at each individual potential glycosylation site were characterized. This information goes for beyond previously published data and sometimes differs from them. During this study, the amino acid sequence originally derived from the DNA sequence of the gene coding for invertase was also verified and it was found that this protein when expressed from SUC2 gene might be created as more than one sequence which differ by a few amino acid substitutions (Asn58<-->Thr, Asn65-->His and Val412<-->Ala).     

Characterization of adducts formed between human serum albumin and the butadiene metabolite epoxybutanediol

1,3-Butadiene (BD) has been classified as a potential human carcinogen. It occurs in the environment as well as in industrial settings. In humans, BD is readily metabolized to reactive epoxides (e.g. 1,2-epoxy-3,4-butanediol). In this study, conjugates between human serum albumin (HSA) and EBD were synthesized (molar ratios of 1:600, 1:1 and 1:0.1; HSA/EBD) under physiological conditions. The 1:600 conjugate and a blank HSA sample were digested with trypsin to obtain specific peptides that were fractionated by preparative liquid chromatography (LC). The fractions were analyzed using nanoelectrospray quadrupole time-of-flight mass spectrometry (nanoES-QqTOFMS). Adducted HSA tryptic peptides were identified and the adducted amino acid residues were identified by sequence analysis based on tandem mass spectrometry (MS/MS). A total of 26 2,3,4-trihydroxybutyl (THB) adducts were identified on 23 tryptic peptides in the HSA/EBD conjugate. The adducted amino acids were the N-terminal aspartic acid residue, six glutamic acid residues, five histidine residues and 14 lysine residues. Results from the nanoES-QqTOFMS experiments were used to set up a more sensitive liquid chromatographic/mass spectrometric (LC/MS) analysis using selected reaction monitoring. Eight of the adducted peptides could be detected in tryptic digests of the 1:0.1 HSA/EBD conjugate.     

A Quantitative Tool to Distinguish Isobaric Leucine and Isoleucine Residues for Mass Spectrometry-Based De Novo Monoclonal Antibody Sequencing

De novo sequencing by mass spectrometry (MS) allows for the determination of the complete amino acid (AA) sequence of a given protein based on the mass difference of detected ions from MS/MS fragmentation spectra. The technique relies on obtaining specific masses that can be attributed to characteristic theoretical masses of AAs. A major limitation of de novo sequencing by MS is the inability to distinguish between the isobaric residues leucine (Leu) and isoleucine (Ile). Incorrect identification of Ile as Leu or vice versa often results in loss of activity in recombinant antibodies. This functional ambiguity is commonly resolved with costly and time-consuming AA mutation and peptide sequencing experiments. Here, we describe a set of orthogonal biochemical protocols, which experimentally determine the identity of Ile or Leu residues in monoclonal antibodies (mAb) based on the selectivity that leucine aminopeptidase shows for n-terminal Leu residues and the cleavage preference for Leu by chymotrypsin. The resulting observations are combined with germline frequencies and incorporated into a logistic regression model, called Predictor for Xle Sites (PXleS) to provide a statistical likelihood for the identity of Leu at an ambiguous site. We demonstrate that PXleS can generate a probability for an Xle site in mAbs with 96% accuracy. The implementation of PXleS precludes the expression of several possible sequences and, therefore, reduces the overall time and resources required to go from spectra generation to a biologically active sequence for a mAb when an Ile or Leu residue is in question.

An algorithm for interpretation of low-energy collision-induced dissociation product ion spectra for de novo sequencing of peptides

An algorithm for interpretation of product ion spectra of peptides generated from ion trap mass spectrometry is developed for de novo amino acid sequencing of peptides for the purpose of protein identification. It is based on a multi-pass analysis of product ion data using a rigorous data extraction and sequence interpretation protocol in the initial pass. The extraction/interpretation algorithm becomes more relaxed in subsequent passes, considering more of the fragment ions, and potentially more sequence candidates. The possible peptide sequences generated by the algorithm are scored according to those sequences which best explain the fragment ion spectrum. These sequences are searched against a protein database using a BLAST search engine to find likely protein candidates. The method is also suitable for locating and determining protein modifications, and can be applied to de novo interpretation of peptide fragment ions in the tandem mass (MS/MS) spectrum produced from a mixture of two peptides having similar nominal mass, but different sequences. Using a known protein, bovine serum albumin, as an example, it is illustrated that this method is rapid and efficient for MS/MS spectral interpretation. This method combined with BLAST programs is then applied to search homologies and to generate information on post-translational modifications of an unknown protein isolated from shark cartilage that does not have a complete genome or proteome database.     

Fast protein sequence determination with matrix-assisted laser desorption and ionization mass spectrometry

Measurement and identification of digested peptides by matrix-assisted laser desorption and ionization mass spectrometry (LDI-MS) is demonstrated. Synthetic human parathyroid hormone, pTH (1-34), with a molecular mass of 4117.8 Da was digested with carboxypeptidases Y and B and the sequence of 14 amino acids from the C-terminus of the peptide was determined by analyzing the molecular mass of the truncated peptides. Furthermore, a tryptic digestion of pTH (1-34) was carried out and a molecular mass map of pTH (1-34) was obtained. With the results of the proteolytic digestion a rapid confirmation of the amino-acid sequence of the protein was possible. It is shown that the results of the tryptic digestion can be used for the unambiguous identification of the amino acid residues Lys and Arg, which cannot be distinguished with a mass spectrometer because of their equal nominal masses. Several advantages of amino acid sequence determination by the combination of digestion and LDI-MS are obvious: high sensitivity in the low pmol range, fast digestion time due to high enzyme/substrate ratios, quantification is unnecessary because the amino acids are identified by their molecular mass differences, the low chemical expenditure for the digestions and the accuracy of the sequence determination. Measurements with LDI-MS are fast: sample preparation and the measurement take only a few min. The mass determination and amino acid sequence is completely unimpaired by amino acid contaminations or impurities in the sample. The sensitivity of the method is in the low pmol to fmol range and thus comparable to other analytical methods.     

Glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the O‐PS of Vibrio cholerae O1, serotype Ogawa,and BSA revealed by matrix‐assisted laser desorption–ionization tandem mass spectrometry

We present the MALDI‐TOF/TOF‐MS analyses of various hapten–bovine serum albumin (BSA) neoglycoconjugates obtained by squaric acid chemistry coupling of the spacer‐equipped, terminal monosaccharide of the O‐specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, to BSA. These analyses allowed not only to calculate the molecular masses of the hapten–BSA neoglycoconjugates with different hapten–BSA ratios (4.3, 6.6 and 13.2) but, more importantly, also to localize the covalent linkages (conjugation sites) between the hapten and the carrier protein. Determination of the site of glycation was based on comparison of the MALDI‐TOF/TOF‐MS analysis of the peptides resulting from the digestion of BSA with similar data resulting from the digestion of BSA glycoconjugates, followed by sequencing by MALDI‐TOF/TOF‐MS/MS of the glycated peptides. The product‐ion scans of the protonated molecules were carried out with a MALDI‐TOF/TOF‐MS/MS tandem mass spectrometer equipped with a high‐collision energy cell. The high‐energy collision‐induced dissociation (CID) spectra afforded product ions formed by fragmentation of the carbohydrate hapten and amino acid sequences conjugated with fragments of the carbohydrate hapten. We were able to identify three conjugation sites on lysine residues (Lys235, Lys437 and Lys455). It was shown that these lysine residues are very reactive and bind lysine specific reagents. We presume that these Lys residues belong to those that are considered to be sterically more accessible on the surface of the tridimensional structure. The identification of the y‐series product ions was very useful for the sequencing of various peptides. The series of a‐ and b‐product ions confirmed the sequence of the conjugated peptides. Copyright © 2010 John Wiley & Sons, Ltd.