Biomedical subjects

Hélène Perreault

Publications and source records attributed to Hélène Perreault.

18 recordsLinked to original sources

PubMed2007

Comparison of the methods for profiling glycoprotein glycans--HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study.

Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.

Carbohydrate Conformation↗

PubMed2006

Use of a 2,5-dihydroxybenzoic acid/aniline MALDI matrix for improved detection and on-target Derivatization of glycans: A preliminary report.

N-Linked glycans derived from human and bovine alpha1-acid glycoprotein, as well as chicken egg white albumin, were analyzed by MALDI-TOF mass spectrometry using a novel MALDI matrix consisting of 2,5-dihydroxybenzoic acid (DHB) and aniline. A significant increase in signal was observed for these oligosaccharides relative to the signal obtained when unmodified DHB was used as a matrix for the same set of samples. The use of aniline/DHB matrix also led to facile on-target derivatization of the glycans via nonreductive amination, as aniline was found to form a stable Schiff base with the reducing end GlcNAc residue without the need for prolonged incubation periods and elevated temperatures. Both native and derivatized glycans ionized as sodium adducts and had similar MS/MS fragmentation patterns consisting mainly of Y/B-cleavage ions. In our experiments, we obtained evidence for persistence of the derivatization reaction in the solid phase; i.e., the reaction appeared to be taking place even after the sample-matrix spot had dried. This is the first report on such solid-phase on-target derivatization of carbohydrates for subsequent analysis by MALDI mass spectrometry.

Albumins↗

PubMed2006

Nonretentive solid-phase extraction of phosphorylated peptides from complex peptide mixtures for detection by matrix-assisted laser desorption/ionization mass spectrometry.

Widespread interest in protein phosphorylation has led to the development of a variety of methods for the analysis of phosphoproteomes of different types of organisms. Many applications involve pretreatment of the sample before mass spectrometric measurement and can crucially improve the detection efficiency of individual phosphopeptides. Despite intense research efforts, separation and extraction of phosphorylated peptides, especially multiphosphorylated ones, remain challenging tasks and need to be further explored and expanded with unconventional approaches. In this study, we describe the application of nonretentive solid-phase extraction (SPE) to the analysis of phosphopeptides using the highly cross-linked polystyrene-divinylbenzene material Strata-X. This study indicates that the procedure allows for the preferential extraction of phosphopeptides regardless of their extent of phosphorylation. The Strata-X material primarily retains nonphosphorylated peptides by hydrophobic interaction, whereas the inherent hydrophilicity of phosphorylated peptides leads to their partitioning into the aqueous phase. Phosphopeptides that were rapidly segregated out of tryptic digest mixtures and collected in the early aqueous fractions generated intense signals in mass spectra. The method was developed using SPE Strata-X columns, then suited for detection and sequencing of phosphopeptides by miniaturizing the system to the scale of custom-made microcolumns. This provided fast isolation of phosphopeptides from protein digests along with direct MALDI on-target deposition. The possibility of on-target washing during sample preparation is also presented.

Amino Acid Sequence↗

PubMed2006

Isolation and identification of sialylated glycopeptides from bovine alpha1-acid glycoprotein by off-line capillary electrophoresis MALDI-TOF mass spectrometry.

Sialylated glycopeptides contained in liquid chromatographic fractions of bovine alpha1-glycoprotein tryptic digests were isolated from asialo peptides using capillary electrophoresis (CE). CE effluents were deposited directly onto a metallic target and analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. This method allowed the characterization of four N-glycosylation sites in the glycoprotein, and each site was observed as a set of sialylated peptide glycoforms. Tandem mass spectrometry was used to confirm peptide sequences and glycan content in glycoforms. The CE method developed for this study resulted in a very clear separation of the sialylated from the asialo content of glycoprotein digests and proved very useful in the determination of the nature and location of sialylated glycans along the protein chain. This article is the first report describing the use of on-target CE fraction collection using a MALDI removable sample concentrator.

Animals↗

PubMed2006

Method for investigation of oligosaccharides from glycopeptides: direct determination of glycosylation sites in proteins.

Characterization of glycopeptides has become an important tool toward a better understanding of the molecular details in carbohydrate-protein interactions. In this approach, oligosaccharides are commonly not detectable under mass spectrometric conditions because of ionization suppression by deglycosylated peptides. Their composition is only deduced from the mass differences between glycopeptides and corresponding deglycosylated peptides. Here, we describe how carbohydrates can be easily detected in the PNGase-treated samples and structurally investigated next to the peptides. The efficacy of this method is demonstrated through the analysis of tryptic glycopeptides obtained from human IgG. Following deglycosylation with PNGaseF and derivatization with phenylhydrazine, MALDI spectra produced ion peaks of labeled oligosaccharides and deglycosylated peptides. The relative abundances of individual oligosaccharides were consistent with those of the glycopeptides. MALDI-MS/MS provided useful data for the structural elucidation of oligosaccharides, including the assignment of dominant isomers and glycosylation sites in peptides. MALDI-MS/MS fragmentation patterns of deglycosylated peptide ions indicated glycosylation sites at asparagine 297 and 299. The observed peptide of the composition ADQTVYR, described for the first time in this study, indicated new glycosylation sites in IgG1 human myeloma plasma.

Chromatography, High Pressure Liquid↗

PubMed2006

Interaction of zonula occludens-1 (ZO-1) with alpha-actinin-4: application of functional proteomics for identification of PDZ domain-associated proteins.

The use of recombinant "bait" proteins to capture protein-binding partners, followed by identification of protein interaction networks by mass spectrometry (MS), has gained popularity and widespread acceptance. We have developed an approach using recombinant PDZ protein interaction modules of the membrane-associated guanylate kinase (MAGUK) protein zonula occludens-1 (ZO-1) to pull-down and screen for proteins that interact with these modules via their PDZ domain binding motifs. Identification of proteins by MS of pull-down material was achieved using a vacuum-based chromatography sample preparation device designed for matrix-assisted laser desorption/ionization (MALDI) MS. MS analysis of tryptic fragments in pull-down material revealed a number of potential ZO-1 interacting candidates, including the presence of peptides corresponding to the cortical membrane scaffolding protein alpha-actinin-4. Interaction of alpha-actinin-4 with ZO-1 was confirmed by coimmunoprecipitation of these two proteins from cultured cells, as well as from brain, liver, and heart, and by immunoblot detection of alpha-actinin-4 after pull-down with the first PDZ domain of ZO-1. In contrast, the highly homologous alpha-actinin family member, alpha-actinin-1, displayed no association with ZO-1. Immunofluorescence showed colocalization of alpha-actinin-4 with ZO-1 in cultured HeLa and C6 glioma cells, as well as in a variety of tissues in vivo, including brain, heart, liver, and lung. This study demonstrates the utility of MS-based functional proteomics for identifying cellular components of the ZO-1 scaffolding network. Our finding of the interaction of ZO-1 with alpha-actinin-4 provides a mechanism for linking the known protein recruitment and signaling activities of ZO-1 with alpha-actinin-4-associated plasma membrane proteins that have regulatory activities at cell-cell and cell-extracellular matrix contacts.

Actinin↗

PubMed2006

N-glycosylation analysis using the StrOligo algorithm.

N-glycosylation of proteins is the predominant glycosylation in mammals and confers specific conformations, localization, and functions to proteins. High-throughput proteomics techniques have focused on the identification of proteins through amino acid sequence determination, with little attention paid to their post-translational modification, in particular, glycosylation. High-throughput mass spectrometric data often contain information about glycosylation, but this is systematically discarded by proteomic search engines. We have developed an algorithm, StrOligo (for STRucture of OLIGOsaccharides), capable of automated analysis of oligosaccharide composition and possible structures by mass spectrometry. The algorithm analyzes tandem mass spectrometry (MS/MS) data in an automated three-step process and provides possible structures and a discrimination score. In the first step, the algorithm constructs a relationship tree of the monosaccharide moiety losses observed in the MS/MS spectrum. In the second step, the algorithm uses the tree to propose possible compositions and structures from combinations of adduct and fragment ions as well as a discrimination score, which reflects the fit with the experimental results. Finally, an interface is available to visualize the proposed structures and their scores. As well, the MS/MS spectrum is displayed with relevant peaks labeled for the proposed structure with the highest discrimination score, using a modified nomenclature.

Algorithms↗

PubMed2005

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.

Algorithms↗

PubMed2005

Influence of the labeling group on ionization and fragmentation of carbohydrates in mass spectrometry.

The ionization and fragmentation behaviors of carbohydrate derivatives prepared by reaction with 2-aminobenzamide (AB), 1-phenyl-3-methyl-5-pyrazolone (PMP), and phenylhydrazine (PHN) were compared under identical mass spectrometric conditions. It has been shown that the intensities of signals in MS spectra depend on the kind of saccharides investigated and reducing end labels used. PMP sialyllactose, when ionized by ESI/MALDI, produced a mixture of [M + H]+, [M + Na]+, [M - H + 2Na]+ ions in the positive mode and [M - H]-, [M + Na - 2H]- ions in the negative mode. The AB and PHN derivatives formed abundant [M + H]+ and [M - H]- ions in ESI, and by matrix-assisted laser desorption/ionization (MALDI) produced abundant [M + Na]+ ions. PMP- and reduced AB-sialyllactose produced only Y-type fragment ions under both MS/MS sources. In the electrospray ionization (ESI)-MS/MS spectrum of PHN-sialyllactose, abundant ions corresponded to B, Z cleavages and in its MALDI-MS/MS spectrum, the abundant ions were consistent with Y glycosidic cleavages with the concurrence of B, C, and cross-ring fragment ions. In the MALDI-MS spectra of oligosaccharides acquired immediately after derivatization, it was possible to detect only PHN derivatives. After purification, spectra of all three types of derivatives showed high signal-to-noise ratios with the most abundant ions observed for AB reduced saccharides. [M + Na]+ ions were the dominant products and their fragmentation patterns were influenced by the type of the labeling and the kind of oligosaccharide considered. In the MALDI-PSD and -MS/MS spectra of AB-derivatized glycans, higher m/z fragment ions corresponded to B and Y cleavages and the loss of bisecting GlcNAc appeared as a weak signal or was not detected at all. Fragmentation patterns observed in the spectra of hybrid/complex PHN and PMP glycans were more comparable-higher m/z fragments corresponded to B and C glycosidic cleavages. For PHN glycans, the abundance of ions resulting from the loss of bisecting GlcNAc depended on the number of residues linked to the 6-positioned mannose. Also, PHN and PMP derivatives produced cross-ring cleavages with abundances higher than observed in the spectra of AB derivatized oligosaccharides. For high-mannose glycans, the most informative cleavages were provided by AB and PHN type of labeling. Here, PMP produced dominant Y-cleavages from the chitobiose while other ions produced weak signals.

Antipyrine↗

PubMed2004

Device for the reversed-phase separation and on-target deposition of peptides incorporating a hydrophobic sample barrier for matrix-assisted laser desorption/ionization mass spectrometry.

The separation of peptide mixtures from proteolytic cleavage is often necessary prior to mass spectrometry (MS) to enhance sensitivity and peptide mapping coverage. When buffers, salts, and other higher abundance peptides/contaminants are present, competition for charge during the electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) processes can lead to ion suppression for the targeted analyte(s). In this note, a simple reversed-phase microcolumn sample separation and deposition device (Sep-Dep) is described. The use of this device improves or renders possible the analysis of complex or contaminated peptide mixtures by MALDI-MS. The method is simple and inexpensive and utilizes single-use low-cost Geloader-type columns packed with reversed-phase material. The device described utilizes an open column, allowing for a gradient or narrow-step gradient to be applied by any solvent delivery system or manually with a pipet. A key feature of the device is a deposition chamber that can be custom-built to hold any MALDI target. The Sep-Dep device is attached directly to an in-house vacuum line and draws solvent from the open-ended LC column. The elution of separated peptides is performed directly onto a target that has been treated with a hydrophobic barrier. This barrier effectively isolates fractions and improves the quality and morphology of the matrix crystals. The method produces efficient separations of proteolytic peptides, significantly reducing signal suppression effects in MALDI.

Amino Acid Sequence↗

PubMed2004

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.

Amino Acid Sequence↗

PubMed2004

Matrix-assisted laser desorption/ionization tandem mass spectrometry and post-source decay fragmentation study of phenylhydrazones of N-linked oligosaccharides from ovalbumin.

N-linked oligosaccharides were released from hen ovalbumin by PNGase F and derivatized with phenylhydrazine. They were then examined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Phenylhydrazones of N-glycans under MALDI-tandem mass spectrometry (MS/MS) and post-source decay (PSD) conditions produced relatively similar fragmentation patterns; however, more cross-ring cleavages and fragment ions corresponding to low abundance isomeric structures were detected by MS/MS and not in PSD. Most fragment ions corresponded to glycosidic cleavages with preferential loss of residues from the chitobiose core and the 3-antenna. Sialylated phenylhydrazone-N-glycans, characterized here for the first time in ovalbumin by tandem mass spectrometry, underwent losses of sialic acid residues followed the same fragmentation pathways observed with neutral derivatized glycans. The relative abundances of some fragment ions indicated the linkage position of sialic acid and provided information on the number of residues attached to the 6-antenna. Also, new structures of ovalbumin glycans were observed as part of this study and are reported here.

Animals↗

PubMed2003

Profiling of N-linked oligosaccharides using phenylhydrazine derivatization and mass spectrometry.

N-linked oligosaccharide standards obtained from commercial sources were derivatized with phenylhydrazine (PHN) and analyzed by on-line reversed-phase high performance liquid chromatography (HPLC)/electrospray ionization mass spectrometry (ESI-MS). This procedure was then applied to mixtures of N-glycans enzymatically released from hen ovalbumin. Under ESI-MS conditions, phenylhydrazones of asialylated oligosaccharide standards and ovalbumin glycans produced mainly [M + 2H]2+ molecular ions at low cone voltage values, while minimal fragmentation was observed. Reversed-phase HPLC/ESI-MS total and selected ion chromatograms obtained for derivatized N-glycans from ovalbumin showed partial but useful separation. Overall glycan profiles obtained by ESI-MS were compared with results obtained by matrix-assisted laser desorption/ionization (MALDI)-MS. Qualitatively, profiles were similar from one technique to the other in terms of relative abundance of glycans versus composition. Post-source decay (PSD) analysis of the [M + Na]+ ions of PHN-glycans showed dominant B, C and internal B/Y, C/Y cleavages. These patterns were helpful in relating fragmentation to proposed structures. Cross-ring cleavage fragment ions (A-type) were also observed in most cases. The PHN derivatization method is fast and simple. It produces abundant parent ions in both MALDI-MS and ESI-MS, while avoiding the presence of salt contaminants during the labeling procedure.

Chromatography, High Pressure Liquid↗

PubMed2003

Labelling saccharides with phenylhydrazine for electrospray and matrix-assisted laser desorption-ionization mass spectrometry.

A well-known reaction of carbonyl compounds with phenylhydrazine has been applied to saccharides, providing increased sensitivity for mass spectrometric (MS) and ultraviolet (UV) detection during high-performance liquid chromatographic (HPLC) separations. After a simple derivatization procedure for 1 h at 70 degrees C and purification of the reaction mixture from excess reagent by extraction, the sugar derivatives were characterized by direct injection or on-line HPLC/electrospray ionization (ESI) and by matrix-assisted laser desorption/ionization (MALDI) MS. Because no salts are used or produced upon reaction, this procedure is very simple and suitable for the tagging of saccharides. The reaction allows for on-target derivatization and products are very stable. The derivatization procedure has been applied to commercially-obtained small saccharides and standard N-linked oligosaccharides. Lastly, hen ovalbumin N-glycans were detached enzymatically and characterized by MALDI-MS as their phenylhydrazone derivatives.

Carbohydrates↗

PubMed2003

Mass spectrometric characterization of proteins from the SARS virus: a preliminary report.

A new coronavirus has been implicated as the causative agent of severe acute respiratory syndrome (SARS). We have used convalescent sera from several SARS patients to detect proteins in the culture supernatants from cells exposed to lavage another SARS patient. The most prominent protein in the supernatant was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a approximately 46-kDa species. This was found to be a novel nucleocapsid protein that matched almost exactly one predicted by an open reading frame in the recently published nucleotide sequence of the same virus isolate (>96% coverage). A second viral protein corresponding to the predicted approximately 139-kDa spike glycoprotein has also been examined by MALDI-TOF MS (42% coverage). After peptide N-glycosidase F digestion, 12 glycosylation sites in this protein were confirmed. The sugars attached to four of the sites were also identified. These results suggest that the nucleocapsid protein is a major immunogen that may be useful for early diagnostics, and that the spike glycoprotein may present a particularly attractive target for prophylactic intervention in combating SARS.

Amino Acid Sequence↗

PubMed2003

Application of the StrOligo algorithm for the automated structure assignment of complex N-linked glycans from glycoproteins using tandem mass spectrometry.

Oligosaccharides associated with proteins are known to give these molecules specific conformations and functions. Analysis of proteins would not be complete without studying the glycans. However, high-throughput techniques in proteomics will soon overwhelm the current capacity of methods if no automation is incorporated into glycomics. New capabilities of the StrOligo algorithm introduced for this purpose (Ethier et al., Rapid Commun. Mass Spectrom., 2002; 16: 1743) will be discussed here. Experimental tandem mass spectra were acquired to test the algorithm using a hybrid quadrupole-time-of-flight (QqTOF) instrument with a matrix-assisted laser desorption/ionization (MALDI) source. The samples were N-linked oligosaccharides from monoclonal antibody IgG, beta interferon and fetuin, detached by enzymatic deglycosylation and labeled at the reducing end. Improvements to the program were made in order to reduce the need for user intervention. StrOligo strips the spectra down to monoisotopic peaks only. The algorithm first builds a relationship tree, accounting for each observed loss of a monosaccharide moiety, and then analyzes the tree and proposes possible structures from combinations of adducts and fragment ion types. A score, which reflects agreement with experimental results, is then given to each proposed structure. The program then decides which combination is the best one and labels relevant peaks in the experimental mass spectrum using a modified nomenclature. The usefulness of the algorithm has been demonstrated by assigning structures to several glycans released from glycoproteins. The analysis was completed in less than 2 minutes for any glycan, which is a substantial improvement over manual interpretation.

Algorithms↗

PubMed2002

A study of immunoglobulin G glycosylation in monoclonal and polyclonal species by electrospray and matrix-assisted laser desorption/ionization mass spectrometry.

N-linked oligosaccharides were released from human and bovine polyclonal immunoglobulin G (IgG) obtained from commercial sources and also from a monoclonal IgG(1) secreted by murine B-lymphocyte hybridoma cells (CC9C10) grown under different serum-free conditions. These conditions differed according to their steady-state dissolved oxygen concentrations. This work is based on a previous quantitative study where released glycans were characterized by fluorophore-assisted carbohydrate electrophoresis (FACE) and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (J. P. Kunkel, D. C. H. Jan, J. C. Jamieson, and M. Butler, 1998, J. Biotechnol. 62, 55-71). In the present article, peptide-N-glycosidase F-released glycans from different species of polyclonal IgG and murine monoclonal IgG were characterized qualitatively by high-performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry (ESI-MS). The glycans were also analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The MALDI mass spectrometer used allowed acquisition of MS and tandem MS data, which were useful in structural investigations at a more detailed level than allowed by FACE and HPAEC-PAD. Predominant N-linked structures, as determined by all techniques, were core-fucosyl asialyl biantennary chains with varying galactosylation. Minor amounts of afucosyl, bisected, and monosialyl oligosaccharides were also detected. In contrast to FACE and HPAEC-PAD, MALDI-double quadrupole/time-of-flight MS and HPLC/ESI-MS also detected low-abundance high-mannose and hybrid structures in some of the species under investigation.

Animals↗

PubMed2002

Automated structural assignment of derivatized complex N-linked oligosaccharides from tandem mass spectra.

Glycoprotein function is controlled by several biological factors, one of them being the structure of carbohydrate chains (glycans) attached to specific amino acids of the protein backbone. Changes in glycan structures have been shown to modify the secondary and tertiary conformation of glycoproteins, thus their function. Powerful analytical tools are available for the characterization of sugar structures, and recently mass spectrometry (MS) has been increasingly useful for this purpose. Manual interpretation of tandem mass spectrum is possible but tedious. Automated interpretation should speed the analysis and enhance the results obtained. A new computer program for automated interpretation of tandem MS spectra of complex N-linked glycans oligosaccharides from mammals will be described. N-Linked oligosaccharides standards were derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) and analyzed by matrix-assisted laser desorption/ionization (MALDI)-tandem MS. Simulated tandem mass spectra of other common glycans were also generated to test the algorithm. The MALDI-MS/MS spectra featured resolved isotopic distributions for the [M + H](+) and fragment ions of oligosaccharides. These isotopic distributions complicated the automated analysis of the spectra and were removed to leave only monoisotopic peaks. An algorithm was written for this purpose, yielding simplified tandem mass spectra. Another algorithm is then used to determine the structure of the oligosaccharide. A score is then given to each structure, depending on agreement with experimental results. The program successfully assigned the true structure in 24 out of the 28 cases (86%) and the true structure was among the three top scoring structures in all cases.

Algorithms↗