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.     

Global and site-specific detection of human integrin alpha 5 beta 1 glycosylation using tandem mass spectrometry and the StrOligo algorithm

Glycans are oligosaccharides associated with proteins, and are known to confer specific functions and conformations on glycoproteins. As protein tridimensional structures are related to function, the study of glycans and their impact on protein folding can provide important information to the field of proteomics. The subdiscipline of glycomics (or glycoproteomics) is rapidly growing in importance as glycans in proteins have shown to be involved in protein-protein or protein-(drug, virus, antibody) interactions. Glycomics studies most often aim at identifying glycosylation sites, and thus are performed on deglycosylated proteins resulting in loss of site-specific details concerning the glycosylation. In order to obtain such details by mass spectrometry (MS), either whole glycoproteins must be digested and analyzed as mixtures of peptides and glycopeptides, or glycans must be isolated from glycopeptide fractions and analyzed as pools. This article describes parallel experiments involving both approaches, designed to take advantage of the StrOligo algorithm functionalities with the aim of characterizing glycosylation microheterogeneity on a specific site. A hybrid quadrupole-quadrupole-time-of-flight (QqTOF) instrument equipped with a matrix-assisted laser desorption/ionization (MALDI) source was used. Glycosylation of alpha 5 beta 1 subunits of human integrin was studied to test the methodology. The sample was divided in two aliquots, and glycans from the first aliquot were released enzymatically, labelled with 2-aminobenzamide, and identified using tandem mass spectrometry (MS/MS) and the StrOligo program. The other aliquot was digested with trypsin and the resulting peptides separated by reversed-phase high-performance liquid chromatography (HPLC). A specific collected fraction was then analyzed by MS before and after glycan release. These spectra allowed, by comparison, detection of a glycopeptide (several glycoforms) and elucidation of peptide sequence. Compositions of glycans present were proposed, and identification of possible glycan structures was conducted using MS/MS and StrOligo.     

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

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

MALDI‐TOF‐Massenspektrometrie: Risikoanalyse im Flug

The high accuracy, molecular resolution and sensitivity of matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) make it an efficient method for analysing all kinds of biomolecules including nucleic acids, proteins/peptides, carbohydrates and lipids. MALDI‐TOF‐MS based high‐throughput genotyping of genetic heterogeneities possesses the potential of becoming a routine method. MAL‐DI‐TOF‐MS can be used for the identification of proteins and posttranslational modifications. Taken together, MALDI‐TOF‐MS represents a integrated platform technology in bioanalytics and molecular medicine.     

Characterization of glycopeptides from HIV-ISF2 gp120 by liquid chromatography mass spectrometry

Previously, we have characterized the HIV-I(SF2) gp120 glycopeptides using matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) and nanospray electrospray ionization (ESI). Although we characterized 25 of 26 consensus glycosylation sites, we could not obtain any information about the extent of sialylation of the complex glycans. Sialylation is known to alter the biological activity of some glycoproteins, e.g., infectivity of some human and nonhuman primate lentiviruses is reduced when the envelope glycoproteins are extensively sialylated, and thus, characterization of the extent of sialylation of complex glycoproteins is of biological interest. Since neither MALDI/MS nor nanospray ESI provided much information about sialylation, probably because of suppression effects inherent in these techniques, we utilized online nanocapillary high performance liquid chromatography (nHPLC) with ESI/MS to characterize the sites and extent of sialylation on gp120. Eight of the known 26 consensus glycosylation sites of HIV-ISF2 gp120 were determined to be sialylated. Two of these sites were previously uncharacterized complex glycans. Thirteen high mannose sites were also determined. The heterogeneity of four of these sites had not been previously characterized. In addition, a peptide containing two consensus glycosylation sites, which had previously been determined to contain complex glycans, was also determined to be high mannose as well.     

Mass spectrometric analysis of the glycosphingolipid‐derived glycans from miniature pig endothelial cells and islets: identification of NeuGc epitope in pig islets

Glycosphingolipid (GSL) is a major component of the plasma membrane in eukaryotic cells that is involved directly in a variety of immunological events via cell‐to‐cell or cell‐to‐protein interactions. In this study, qualitative and quantitative analyses of GSL‐derived glycans on endothelial cells and islets from a miniature pig were performed and their glycosylation patterns were compared. A total of 60 and 47 sialylated and neutral GSL‐derived glycans from the endothelial cells and islets, respectively, were characterized by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and collision‐induced fragmentation using positive‐ion electrospray ionization (ESI) ion‐trap tandem mass spectrometry (MS/MS). In accordance with previous immunohistochemistry studies, the α‐Gal‐terminated GSL was not detected but NeuGc‐terminated GSLs were newly detected from miniature pig islets. In addition, the neutral GSL‐derived glycans were relatively quantified by derivatization with carboxymethyl trimethylammonium hydrazide (so called Girard's T reagent) and MALDI‐TOF MS. The structural information of the GSL‐derived glycans from pig endothelial cells and islets suggests that special attention should be paid to all types of glycoconjugates expressed on pig tissues or cells for successful clinical xenotransplantation. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of N-glycans of recombinant human thyrotropin using mass spectrometry

Thyroid-stimulating hormone is a vital component of the regulatory mechanism that maintains the structure and function of the thyroid gland and governs thyroid hormone release. In this paper we report the first detailed structural characterization of the N-linked oligosaccharides of recombinant human thyroid-stimulating hormone (rhTSH). Using a strategy combining mass spectrometric analysis and sequential exoglycosidase digestion, we have defined the structures of the N-glycans released from recombinant human thyrotropin by peptide N-glycosidase F. All glycans are complex-type glycans and are mainly of the bi- and triantennary type with variable degrees of fucosylation and sialylation. The major non-reducing epitope in the complex-type glycans is: NeuAcalpha2-3Galbeta1-4GlcNAc (sialylated LacNAc). The carbohydrate microheterogeneity at the three glycosylation sites was studied using reversed-phase high-performance liquid chromatography (RP-HPLC), concanavalin A affinity chromatography and mass spectrometric techniques, including both matrix-assisted laser desorption/ionization (MALDI) and electrospray. rhTSH was reduced, carboxymethylated and then digested with trypsin. The mixture of peptides and glycopeptides was subjected to RP-HPLC and the structures of the glycopeptides were determined by MALDI in conjunction with on-target exoglycosidase digestions. After PNGase F digestion, the peptide moiety of the glycopeptide was determined by the presence of the b- and y-series ions derived from its amino acid sequence in the quadrupole time-of-flight tandem mass (QTOF-MS/MS) spectrum. Glycosylation sites Asn-alpha52 and Asn-alpha78 contain mainly bi- and triantennary complex-type glycans. Only glycosylation site Asn-alpha52 bears fucosylated N-glycans. Minor tetraantennary complex structures were also observed on both glycosylation sites. Profiling of the carbohydrate moieties of Asn-beta23 indicates a large heterogeneity. Bi-, tri-, and tetraantennary N-glycans were present at this site. These data demonstrate site-specificity of glycosylation in the alpha subunit but not in the beta subunit of rhTSH with Asn-alpha52 bearing essentially di- and triantennary glycans with or without core fucosylation and bi- and triantennary glycans with no core fucosylation being attached to Asn-alpha78.     

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.     

MSQ: a tool for quantification of proteomics data generated by a liquid chromatography/matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry based targeted quantitative proteomics platform

Mass spectrometry (MS)‐based quantitative proteomics has become a critical component of biological and clinical research for identification of biomarkers that can be used for early detection of diseases. In particular, MS‐based targeted quantitative proteomics has been recently developed for the detection and validation of biomarker candidates in complex biological samples. In such approaches, synthetic reference peptides that are the stable isotope labeled version of proteotypic peptides of proteins to be quantitated are used as internal standards enabling specific identification and absolute quantification of targeted peptides. The quantification of targeted peptides is achieved using the intensity ratio of a native peptide to the corresponding reference peptide whose spike‐in amount is known. However, a manual calculation of the ratios can be time‐consuming and labor‐intensive, especially when the number of peptides to be tested is large. To establish a liquid chromatography/matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry (LC/MALDI TOF/TOF)‐based targeted quantitative proteomics pipeline, we have developed a software named Mass Spectrometry based Quantification (MSQ). This software can be used to automate the quantification and identification of targeted peptides/proteins by the MALDI TOF/TOF platform. MSQ was applied to the detection of a selected group of targeted peptides in pooled human cerebrospinal spinal fluid (CSF) from patients with Alzheimer's disease (AD) in comparison with age‐matched control (OC). The results for the automated quantification and identification of targeted peptides/proteins in CSF were in good agreement with results calculated manually. Copyright © 2010 John Wiley & Sons, Ltd.     

Selective enrichment of N‐linked glycopeptides and glycans by using a dextran‐modified hydrophilic material

Glycosylation analysis of proteins from biological sources utilizing mass spectrometry based approaches is challenging due to the relatively low abundance of glycopeptides, the structural diversity of glycans, and the coexisting matrices. In this study, a customized dextran‐bonded silica‐based stationary phase was introduced for selective enrichment of glycopeptides and glycans from complex biological samples. This material has exhibited superior selectivity and broader glycosylation site coverage over commercial Sepharose in glycoproteomic evaluation. Additionally, the glycomic analysis of fetuin, α₁‐acid glycoprotein, and human serum N‐glycome also indicated the relatively higher sensitivity, selectivity, and glycoform coverage of dextran‐bonded silica than that of Sepharose and porous graphitized carbon. Therefore, the dextran‐bonded silica is expected to make contributions in the fields of glycoproteomics and glycomics.     

Analysis of monoclonal antibody by a novel CE‐UV/MALDI‐MS interface

mAbs are highly complex proteins that present a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product‐ and time‐consuming. CE‐MS couplings, especially to MALDI, appear really attractive methods for the characterization of biological samples. In this work, we report the last instrumental development and performance of the first totally automated off‐line CE‐UV/MALDI‐MS/MS. This interface is based on the removal of the original UV cell of the CE apparatus, modification of the spotting device geometry, and creation of an integrated delivery matrix system. The performance of the method was evaluated with separation of five intact proteins and a tryptic digest mixture of nine proteins. Intact protein application shows the acquisition of electropherograms with high resolution and high repeatability. In the peptide mapping approach, a total number of 154 unique identified peptides were characterized using MS/MS spectra corresponding to average sequence coverage of 64.1%. Comparison with NanoLC/MALDI‐MS/MS showed complementarity at the peptide level with an increase of 42% when using CE/MALDI‐MS coupling. Finally, this work represents the first analysis of intact mAb charge variants by CZE using an MS detection. Moreover, using a peptide mapping approach CE‐UV/MALDI‐MS/MS fragmentation allowed 100% sequence coverage of the light chain and 92% of the heavy chain, and the separation of four major glycosylated peptides and their structural characterization.     

Targeted glycoproteomics: serial lectin affinity chromatography in the selection of O-glycosylation sites on proteins from the human blood proteome

Although lectin selection is gaining increasing acceptance as a tool for targeting glycosylation in glycoproteomics, most of the work has been directed at N-glycosylation. The work reported here focuses on the use of lectins in the study of O-glycosylation. The problem with using lectins for studying O-glycosylation is that they are not sufficiently specific. This paper reports that through the use of serial lectin affinity chromatography (SLAC) it is possible to select predominantly O-glycosylated peptides from tryptic digests of human serum. Jacalin is relatively specific for O-glycosylation but has the problem that it also selects high mannose N-type glycans. This problem was addressed by using a concanavalin A affinity column to first remove high mannose, hybrid-type and biantennary complex-type N-type glycans before application of the Jacalin columns. When used in a serial format, concanavalin A and Jacalin together provide essentially O-glycosylated peptides. The glycoprotein parents of glycopeptides were identified by deglycosylating the selected O-glycopeptides by oxidative elimination. These peptides were then separated by RPC and further analyzed using ESI-MS/MS and MALDI-MS/MS. Using this approach all the O-glycosylated sites in a model protein (fetuin) and over thirty glycoprotein parents from human serum were identified. It is concluded that a serial combination of Con A and Jacalin can be of utility in the study of O-glycosylation in glycoproteomics.     

Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS

Site-specific characterisation of mucin-type O-linked glycosylation is an analytical challenge due to glycan heterogeneity, lack of glycosylation site consensus sequence and high density of occupied glycosylation sites. Here, we report the use of electron transfer dissociation (ETD) for the site-specific characterisation of densely glycosylated mucin-type O-linked glycopeptides using ESI-IT-MS/MS. Synthetic glycopeptides from the human mucin-1 (MUC-1) tandem repeat region containing a range of O-linked, tumour-associated carbohydrate antigens, namely Tn, T and sialyl T, with different glycosylation site occupancies and an increasing number of tandem repeats were studied. In addition, a glycopeptide from the anti-freeze glycoprotein of Antarctic and Arctic notothenoids, bearing four O-linked, per-acetylated T antigens was characterised. ETD MS/MS of infused or capillary LC-separated glycopeptides provided broad peptide sequence coverage (c/z·-type fragment ions) with intact glycans still attached to the Ser/Thr residues. Thus, the glycosylation sites were unambiguously determined, while simultaneously obtaining information about the attached glycan mass and peptide identity. Highly sialylated O-glycopeptides showed less efficient peptide fragmentation, but some sequence and glycosylation site information was still obtained. This study demonstrates the capabilities of ETD MS/MS for site-specific characterisation of mucin-type glycopeptides containing high-density O-linked glycan clusters, using accessible and relative low-resolution/low-mass accuracy IT MS instrumentation.     

Derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate for enhancing the ionization yield of small peptides and glycopeptides in matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

The characterization of glycosylation in proteins by mass spectrometry (MS) is often impeded by strong suppression of ionization of glycopeptides in the presence of non-glycosylated peptides. Glycopeptides with a large carbohydrate part and a short peptide backbone are particularly affected by this problem. To meet the goal of generating mass spectra exhibiting glycopeptide coverages as complete as possible, derivatization of glycopeptides offers a practical way to increase their ionization yield. This paper investigated derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) which is a rapid labeling technique commonly used for fluorescence detection in high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). As test samples we used peptides and glycopeptides obtained by enzymatic digestion of three different glycoproteins, i.e., human antithrombin, chicken ovalbumin, and bovine alpha1-acid-glycoprotein. It was found that AQC derivatization resulted in strongly increased signal intensities when analyzing small peptides and glycopeptides by matrix-assisted laser desorption/ionization (MALDI)-MS. For these compounds the limit of detection could be reduced to low fmol amounts. Without derivatization only glycopeptides containing large peptide backbones were detected by MALDI-MS. This effect was even significant when glycopeptides were pre-separated and enriched by means of lectin affinity chromatography before MALDI-MS analysis and when using electrospray ionization (ESI). This labeling method, applied in combination with MS detection for the first time, was found to be well suited for the enhancement of detection sensitivity for small glycopeptides in MALDI-MS analysis and thus for reducing the need for pre-separation steps.     

Qualitative and quantitative comparison of N‐glycans between pig endothelial and islet cells by high‐performance liquid chromatography and mass spectrometry‐based strategy

N‐glycan structures released from miniature pig endothelial and islet cells were determined by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF), negative ion electrospray ionization (ESI) MS/MS and normal‐phase high performance liquid chromatography (NP‐HPLC) combined with exoglycosidase digestion. Totally, the identified structures were 181 N‐glycans including 129 sialylated and 18 α‐galactosylated glycans from pig endothelial cells and 80 N‐glycans including 41 sialylated and one α‐galactosylated glycans from pig islet cells. The quantity of the α‐galactosylated glycans from pig islet cells was certainly neglectable compared to pig endothelial cells. A number of NeuGc‐terminated N‐glycans (80 from pig endothelial cells and 13 from pig islet cells) are newly detected by our mass spectrometric strategies. The detailed structural information will be a matter of great interest in organ or cell xenotransplantation using α 1,3‐galactosyltransferase gene‐knockout (GalT‐KO) pig. Copyright © 2009 John Wiley & Sons, Ltd.     

A procedure for the analysis of site‐specific and structure‐specific fucosylation in alpha‐1‐antitrypsin

A MS‐based methodology has been developed for analysis of core‐fucosylated versus antennary‐fucosylated glycosites in glycoproteins. This procedure is applied to the glycoprotein alpha‐1‐antitrypsin (A1AT), which contains both core‐ and antennary‐fucosylated glycosites. The workflow involves digestion of intact glycoproteins into glycopeptides, followed by double digestion with sialidase and galactosidase. The resulting glycopeptides with truncated glycans were separated using an off‐line HILIC (hydrophilic interaction liquid chromatography) separation where multiple fractions were collected at various time intervals. The glycopeptides in each fraction were treated with PNGase F and then divided into halves. One half of the sample was applied for peptide identification while the other half was processed for glycan analysis by derivatizing with a meladrazine reagent followed by MS analysis. This procedure provided site‐specific identification of glycosylation sites and the ability to distinguish core fucosylation and antennary fucosylation via a double digestion and a mass profile scan. Both core and antennary fucosylation are shown to be present on various glycosites in A1AT.     

On‐chip solid‐phase extraction pre‐concentration/focusing substrates coupled to atmospheric pressure matrix‐assisted laser desorption/ionization ion trap mass spectrometry for high sensitivity biomolecule analysis

Atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI) has proven a convenient and rapid method for ion production in the mass spectrometric (MS) analysis of biomolecules. AP‐MALDI and electrospray ionization (ESI) sources are easily interchangeable in most mass spectrometers. However, AP‐MALDI suffers from less‐than‐optimal sensitivity due to ion losses during transport from the atmosphere into the vacuum of the mass spectrometer. Here, we study the signal‐to‐noise ratio (S/N) gains observed when an on‐chip dynamic pre‐concentration/focusing approach is coupled to AP‐MALDI for the MS analysis of neuropeptides and protein digests. It was found that, in comparison with conventional AP‐MALDI targets, focusing targets showed (1) a sensitivity enhancement of approximately two orders of magnitude with S/N gains of 200–900 for hydrophobic substrates, and 150–400 for weak cation‐exchange (WCX) substrates; (2) improved detection limits as low as 5 fmol/µL for standard peptides; (3) significantly reduced matrix background; and (4) higher inter‐day reproducibility. The improved sensitivity allowed successful tandem mass spectrometric (MS/MS) sequencing of dilute solutions of a derivatized tryptic digest of a protein standard, and enabled the first reported AP‐MALDI MS detection of neuropeptides from Aedes aegypti mosquito heads. Copyright © 2009 John Wiley & Sons, Ltd.     

Site-specific N-glycosylation analysis: matrix-assisted laser desorption/ionization quadrupole-quadrupole time-of-flight tandem mass spectral signatures for recognition and identification of glycopeptides

The identification of glycosylation sites in proteins is often possible through a combination of proteolytic digestion, separation, mass spectrometry (MS) and tandem MS (MS/MS). Liquid chromatography (LC) in combination with MS/MS has been a reliable method for detecting glycopeptides in digestion mixtures, and for assigning glycosylation sites and glycopeptide sequences. Direct interfacing of LC with MS relies on electrospray ionization, which produces ions with two, three or four charges for most proteolytic peptides and glycopeptides. MS/MS spectra of such glycopeptide ions often lead to ambiguous interpretation if deconvolution to the singly charged level is not used. In contrast, the matrix-assisted laser desorption/ionization (MALDI) technique usually produces singly charged peptide and glycopeptide ions. These ions require an extended m/z range, as provided by the quadrupole-quadrupole time-of-flight (QqTOF) instrument used in these experiments, but the main advantages of studying singly charged ions are the simplicity and consistency of the MS/MS spectra. A first aim of the present study is to develop methods to recognize and use glycopeptide [M+H]+ ions as precursors for MS/MS, and thus for glycopeptide/glycoprotein identification as part of wider proteomics studies. Secondly, this article aims at demonstrating the usefulness of MALDI-MS/MS spectra of N-glycopeptides. Mixtures of diverse types of proteins, obtained commercially, were prepared and subjected to reduction, alkylation and tryptic digestion. Micro-column reversed-phase separation allowed deposition of several fractions on MALDI plates, followed by MS and MS/MS analysis of all peptides. Glycopeptide fractions were identified after MS by their specific m/z spacing patterns (162, 203, 291 u) between glycoforms, and then analyzed by MS/MS. In most cases, MS/MS spectra of [M+H]+ ions of glycopeptides featured peaks useful for determining sugar composition, peptide sequence, and thus probable glycosylation site. Peptide-related product ions could be used in database search procedures and allowed the identification of the glycoproteins.     

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.     

Protein identification by accurate mass matrix-assisted laser desorption/ionization imaging of tryptic peptides

The spatial distribution of proteins in tissue sections can be used to identify potential markers for pathological processes. Tissue sections are often subjected to enzymatic digestion before matrix‐assisted laser desorption/ionization (MALDI) imaging. This study is targeted at improving the on‐tissue identification of tryptic peptides by accurate mass measurements and complementary off‐line liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) analysis. Two adjacent mouse brain sections were analyzed in parallel. The first section was spotted with trypsin and analyzed by MALDI imaging. Direct on‐tissue MS/MS experiments of this section resulted in the identification of 14 peptides (originating from 4 proteins). The second tissue section was homogenized, fractionated by ultracentrifugation and digested with trypsin prior to LC/ESI‐MS/MS analysis. The number of identified peptides was increased to 153 (corresponding to 106 proteins) by matching imaged mass peaks to peptides which were identified in these LC/ESI‐MS/MS experiments. All results (including MALDI imaging data) were based on accurate mass measurements (RMS <2 ppm) and allow a confident identification of tryptic peptides. Measurements based on lower accuracy would have led to ambiguous or misleading results. MS images of identified peptides were generated with a bin width (mass range used for image generation) of Δm/z = 0.01. The application of accurate mass measurements and additional LC/MS measurements increased both the quality and the number of peptide identifications. The advantages of this approach for the analysis of biological tissue sections are demonstrated and discussed in detail. Results indicate that accurate mass measurements are needed for confident identification and specific image generation of tryptic peptides in tissue sections. Copyright © 2011 John Wiley & Sons, Ltd.