Characterization Of Glycopeptides Research Articles

Electron Capture Dissociation of Triantennary Complex-Type N-Glycosylated Peptides: A Case of Suppressed Peptide Backbone Cleavage

We investigated the electron capture dissociation (ECD) of triantennary complex-type N-glycosylated peptides prepared from bovine fetuin. Previous reports on Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) suggested that ECD is an advantageous method for structural characterization of glycopeptides, because it can cleave the N-Cα bond on the peptide backbone while retaining the labile glycosidic bonds. We present here new data from ECD which resulted in the degradation of the glycan structure prior to any backbone cleavage. Based on the ECD experiment on deglycosylated samples, the character of the peptide backbone sequence, which contains carbamoylmethylated cysteine residue, seemed to prevent extensive N-Cα bond cleavage. It appears that more basic investigations are necessary to promote practical use of ECD for structural characterization of complex glycopeptides because each type of glycopeptide exhibits particular fragmentation patterns.

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.

Structural Characterization of Glycopeptides by N-terminal Protein Ladder Sequencing

High-sensitivity and high-throughput mass spectrometry (MS) has become an important tool for characterizing glycopeptides. Here, we analyzed synthetic O-linked glycopeptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. First, we applied MALDI-quadrupole ion trap (QIT)-TOF MS, which enables collision-induced dissociation-MSn analysis for fine structural characterization. Subsequent MS/MS of sodium adduct ions selected as precursor ions yielded detailed information about the site of oligosaccharide attachment as well as the carbohydrate and amino acid sequences; however, these MS/MS spectra were very complex. To obtain easily interpretable and simple spectra, we used N-terminal protein ladder sequencing coupled with MALDI-TOF MS. From the extremely simple resulting spectra, we were able to determine the glycosylation sites, amino acid sequences, and oligosaccharide molecular weights of the glycopeptides.

A Novel Approach for Identification and Characterization of Glycoproteins Using a Hybrid Linear Ion Trap/FT-ICR Mass Spectrometer

Combining source collision-induced dissociation (CID) and tandem mass spectral acquisition in a pseudo-MS(3) experiment using a linear ion trap results in a highly selective and sensitive approach to identifying glycopeptide elution from a protein digest. The increased sensitivity is partially attributed to the nonselective nature of source CID, which allows simultaneous activation of all charge states and coeluting glycoforms generating greater ion abundance for the mass-to-charge (m/z) 204 and/or 366 oxonium ions. Unlike source CID alone, a pseudo-MS(3) approach adds selectivity while improving sensitivity by eliminating chemical noise during the tandem mass spectral acquisition of the oxonium ions in the linear ion trap. Performing the experiments in the hybrid linear ion trap/Fourier transform-ion cyclotron resonance (FT-ICR) enables subsequent high-resolution/high-mass accuracy full-scan mass spectra (MS) and parallel acquisition of MS/MS in the linear ion trap to be completed in 2 s directly following the pseudo-MS(3) scan to collate identification and characterization of glycopeptides in one experimental scan cycle. Analysis of bovine fetuin digest using the combined pseudo-MS(3), high-resolution MS, and data-dependent MS/MS events resulted in identification of four N-linked and two O-linked glycopeptides without enzymatic cleavage of the sugar moiety or release of the sialic acids before analysis. In addition, over 95% of the total protein sequence was identified in one analytical run.

Characterization of glycopeptides from HIV-I SF2 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.

The information encrypted in accurate peptide masses-improved protein identification and assistance in glycopeptide identification and characterization.

Analytically useful information from accurate mass data for peptides with an error of </=20 ppm is discussed. The deltamass (= mass value following the decimal point) distribution of natural peptides is extracted from a protein database. Compared with the random peptide data, the natural data show a higher average deltamass value and a smaller width of the mass distribution. This deviation can be ascribed to the non-random abundances of the standard amino acids. In particular, accurate mass data for peptides located near the edges of the natural mass distribution contain analytical information. Mass data near the edges generate very few hits in a protein database search and are therefore highly specific for protein identification. Mass signals near the low-mass edge indicate either a high probability that the peptide contains one or several cysteine sites, or that the peptide is highly acidic due to the presence of several D and/or E residues or that it is a glycopeptide. Mass data near the high-mass edge indicate a non-polar peptide with a high abundance of the non-polar amino acids leucine, isoleucine and valine. An Internet page is introduced that analyzes the deviation of a peptide mass from the average deltamass value and that supports the characterization of glycopeptides found near the low-mass edge of the mass distribution.

6] Identification and characterization of glycopeptides in tryptic maps by high-pH anion-exchange chromatography

This chapter describes the application of high-pH anion-exchange chromatography, with pulsed amperometric detection (HPAEC-PAD) to the identification and partial characterization of glycopeptides, within a reversed-phase high-performance liquid chromatography (HPLC) peptide map. HPAEC is a versatile chromatographic method that can be used for the detection and quantitation of monosaccharides, released by acid hydrolysis and for the analysis of intact oligosaccharides released enzymatically or chemically. A large number of other analytical methods, including mass spectrometry (MS), enzymatic microsequencing, nuclear magnetic resonance (NMR), and capillary electrophoresis, can also be applied to the characterization of glycopeptides; in general, a combination of several complementary analytical methods are needed to achieve a definitive carbohydrate structure determination. This chapter discusses the type of information that can be obtained, using a single, comparatively simple, analytical method.

Characterization of Glycopeptides from Recombinant Coagulation Factor VIIa by High-Performance Liquid Chromatography and Capillary Zone Electrophoresis Using Ultraviolet and Pulsed Electrochemical Detection

Four glycopeptide (GP) fractions containing glycosylated Asn 322 were isolated from a tryptic digest of recombinant coagulation factor VII by reversed-phase-HPLC (RP-HPLC). Characterization of the GPs by enzymatic desialylation and RP-HPLC as well as by enzymatic deglycosylation, RP-HPLC, and high-pH anion-exchange chromatography indicated that the four GPs consisted of the same decapeptide but with 0, 1, 2, or 4 residues of sialic acid. In comparison to HPLC, capillary zone electrophoresis (CZE) using uv and pulsed electrochemical detection (PED) afforded improved separation of GPs from each other and from contaminants. CZE-uv and CZE-PED of the desialylated GPs and deglycosylated GPs corroborated the results obtained with the chromatographic methods.

Identification, Quantification, and Characterization of Glycopeptides in Reversed-Phase HPLC Separations of Glycoprotein Proteolytic Digests

Monosaccharide analysis by high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD) was used to identify the glycopeptides in the reversed-phase (RP-HPLC) separation of a bovine fetuin tryptic digest (1.6 nmol). This method, requiring no sample derivatization, identified four asparagine-linked (N-linked) glycopeptides and at least seven serine/threonine-linked (O-linked) glycopeptides. Glycopeptide identification was confirmed by Edman sequencing. Monosaccharide quantification of each glycopeptide suggested that all of the N-linked glycopeptides were the complex type and all the O-linked glycopeptides were sialylated. We determined that glycopeptides could be prepared by acidic reversed-phase chromatography with less than 3% loss of N-acetylneuraminic acid (Neu5Ac). The N-linked glycopeptides of bovine fetuin were prepared, digested with N-glycosidase F (PNGase F), and their oligosaccharides analyzed by HPAEC/PAD. These oligosaccharide profiles revealed that the Asn-138 oligosaccharide attachment site contained the majority of the disialylated and monosialylated oligosaccharides. The Asn-158 oligosaccharide attachment site contained the majority of the tetrasialylated oligosaccharides.

Characterization of glycopeptides from Physarum polycephalum labeled with (3H)mannose or (3H)glucosamine.

Glycopeptides from the microplasmodia of Physarum polycephalum grown in the presence of [3H]mannose or [3H]glucosamine were characterized by gel-filtration, anion-exchange and lectin-affinity chromatography before and after enzymatic or chemical treatment. Various types of glycopeptide were found in the growing plasmodia as shown by Con A affinity column chromatography, while pulse-labeled precursors were composed mainly of mannose-rich glycopeptides which were sensitive to endo H and α-mannosidase. During the processing of the glycoprotein, the mannose-rich glycopeptides changed to larger glycopeptides which were resistant to endo H and α-mannosidase. These processing reactions of the glycoprotein appeared to be blocked when the plasmodia underwent differentiation to macrocysts.

Characterization of human fibronectin glycopeptides from cystic fibrosis and control skin fibroblasts

Fibronectins isolated from different species and tissue sources are glycosylated differently. We report here a characterization of the glycopeptides of fibronectin isolated from the culture medium of skin fibroblasts from patients with cystic fibrosis together with age-, race-, and sex-matched control subjects. The characterization of this fibronectin is of special interest because it is derived from: ( 1) a non-fetal, cellular source; ( 2) eight different individuals; and ( 3) cystic fibrosis and control individuals. The fibronectin glycopeptides were purified by gel-permeation chromatography and Con A-Sepharose and were analyzed by anion-exchange chromatography and affinity columns of immobilized 5-hydroxytryptamine and lectins. One half of the glycopeptides of skin fibroblast fibronectin were shown to contain biantennary oligosaccharides which were core-fucosylated and partially sialylated. Although the remaining half was a complex mixture of glycopeptides, there was remarkably little inter-individual variation. No difference between cystic fibrosis and control subjects was discernible by the techniques employed here. Unlike the biantennary glycopeptides of human plasma fibronectin, those from skin fibroblast fibronectin were core-fucosylated and less highly sialylated. However, compared to human cellular fibronectin glycopeptides from fetal sources, those from skin fibroblast fibronectin were both more highly fucosylated and sialylated.

Isolation and characterization of glycopeptides of human transcortin

Pronase digestion of human transcortin yielded two major glycopeptides (G1D3 and G2D2, in a molar ratio of approx. 1.0 to 1.5) which were isolated by gel and anion-exchange chromatography. Chromatography behaviour, methylation analysis and analytical chromatography on a Con A-Sepharose column suggested that both of the gtycopeptides were N-linked asparaginyl oligosaccharides of the N-acetytiactosamine type, G1D3 and G2D2 being triantennary and biantennary isoglycans, respectively. Microheterogeneity in regard to the position of the linkages between sialyl and galactosyl residues occurred in the triantennary and, possibly, in biantennary isoglycans.

Studies on carbohydrate moieties ofAspergillus niger glucoamylase II: Isolation, purification and characterization of glycopeptides

Six glycopeptide fractions namely GP-C1, GP-C2. GP-C3a.GP-C3b.GP-D, and GP-D2 were isolated after exhaustive digestion of glucoamylase II (Glucozyme) fromAspergillus niger with pronase. They were purified using gel-filtration. high-voltage paper electrophoresis and ion-exchange chromatography on Dowex-50 and Dowex-1. They appeared homogeneous on electrophoresis under different conditions of pHs. The molecular weights ranged from 1600 and 4000 for these glycopeptides. Ally of them contained serine at the N-terminal end. Serine and threonine were the major amino acids with glycine, alanine, proline and tryosine present as minor constituents. Carbohydrate analysis revealed the presence of different sugars. Based on this, the glycopeptides were grouped into three types: (1) GP-C1 and GP-C2 containing mannose, glucose and galactose; (2) GP-C3a, and GP-C3b,containing mannose glucose and glucosamine; and (3) GP-D1 and GP-D2, containing mannose. glucose, galactose and xylose. Most sugar constituents in each glycopeptide occured in non-integral ratios implying a microheterogeneity of the carbohydrate moiety inAspergillus niger glucoamylase.

Characterization of glycopeptides labelled from D-[2-3H]mannose and L-[6-3H]fucose in intestinal epithelial cell membranes during differentiation.

The labelled glycopeptides obtained by Pronase digestion of rat intestinal epithelial cell membranes were examined by gel filtration after injection of D-[2-3H]mannose and L-[6-3H]fucose. Three labelled fraction were eluted in the following order from Bio-Gel P-6, Fraction I, which was excluded from the gel, was labelled mostly with [3H]fucose and slightly with [3H]mannose. Fraction II contained "complex" asparagine-linked oligosaccharides since it was labelled with [3H]mannose and [3H]fucose, was stable to mild alkali treatment, and resistant to endo-beta-N-acetyl-glucosaminidase H. Fraction III contained "high-mannose" asparagine-linked oligosaccharides, which were labelled with [3H]mannose, but not with [3H]fucose; these were sensitive to endo-beta-N-acetylglucosaminidase H, and were adsorbed on concanavalin A-Sepharose and subsequently eluted with methyl alpha-D-mannopyranoside. The time course of incorporation of [3H]mannose into these glycopeptides in microsomal fractions showed that high-mannose oligosaccharides were precursors of complex oligosaccharides. The rate of this processing was faster in rapidly dividing crypt cells than in differentiated villus cells. The ratio of radioactively labelled complex oligosaccharides to high-mannose oligosaccharides, 3h after [3H]mannose injection, was greater in crypt than in villus-cell lateral membranes. Luminal membranes of both crypt and villus cells were greatly enriched in labelled complex oligosaccharides compared with the labelling in lateral-basal membranes. These studies show that intestinal epithelial cells are polarized with respect to the structure of the asparagine-linked oligosaccharides on their membrane glycoproteins. During differentiation of these cells quantitative differences in labelled membrane glycopeptides, But no major qualitative change, were observed.

Preliminary characterization of glycopeptides derived from glycoproteins specified by the Flavivirus Kunjin

The oligosaccharide components of the glycoproteins observed in Vero cells infected by the flavivirus Kunjin were separated by gel filtration following exhaustive Pronase digestion of radioactively labeled glycoproteins. The glycopeptides of gp59, gp56, gp53, and NV2 fell into four classes based on size: (i) 3600 to 4200 daltons, (ii) 2800 to 3100 daltons, (iii) 2300 to 2500 daltons, and (iv) 1500 to 1700 daltons. Glycopeptides of the first class were detected in gp59 and gp56, of the second in gp59, gp56, and gp53, and of the third and fourth classes in gp59, gp53, and NV2. Complex oligosaccharides containing fucose, galactose, mannose, and glucosamine were found in the first two classes and possibly in the third. Oligosaccharides in the smallest class were labeled with [ 3H]mannose and [ 3H]glucosamine only.

Characterization of glycopeptides isolated from membranes of F9 embryonal carcinoma cells

From cells of a nullipotential line of embyonal carcinoma was isolated a membrane fraction enriched in the cell surface F9 antigen. More than 40% fo the radioactive fucose and galactose incorporated by cells into nondialyzable material was recovered in this membrane preparation, corresponding to an aproximately 10-fold purification of the labeled material. Expreme heterogeneity of membrane of glycoproteins labeled wth these sugars was revealed by sodium dodecyl sulfate gel electrophoresis. Glycopeptides prepared by extensive pronase digestion of membranes labeled with fucose or galactose showed properties similar to those already described for fucose-labeled glycopeptides from whole cells. Namely, large glycopeptides eluted near the excluded volume of Sephadex G-50 colum were the predominant glycopeptide species, while complex clycopeptides of molecular weight aroung 2500 were minor components. Therefore, these largely glycopeptides, characteristics of embryonal carcinoma cells, are derived mainly from a variety of glycoproteins closely associated with the membrane system, most probably cell-surface membrane of the cells. The large glycopeptides were also significantly labeled with glucosamine, but only slight with mannose; major components of mannose-labeled glycopeptides from the membranes were high-mannose glycopeptides of low molecular weight. Several experimients excluded gthe possibility that the large glycopeptides are mucopolysaccharides, glycolipids or mucin-tye glycoproteins with short oligosaccharide chains.

Carbohydrate of the human plasminogen variants. III. Structure of the O-glycosidically linked oligosaccharide unit.

The preceding two manuscripts in this issue (Hayes, M. L., and Castellino, F. J. (1979) J. Biol. Chem. 254, 8768-8771, 8772-8776) describe the isolation and characterization of glycopeptides from human plasminogen affinity chromatography variants 1 and 2. Plasminogen variant 1 contains an asparagine288-based branched carbohydrate structure, which has been established in the immediately preceding manuscript. This structure is absent in variant 2. Plasminogen variants 1 and 2 contain a threonine-based glycoconjugate. This latter structure has been established by combination of methylation data, glycosidase digestions, periodate oxidations, and Smith degradations of the beta-eliminated reduced oligosaccharides. One glycopeptide unit, isolated from both plasminogen variants 1 (1D) and 2 (2D) possessed the following structure: Sia alpha 2 yields 3Gal beta 1 yields 3GalNAc-Thr. The threonine was found to be residue 345 in the Glu-plasminogen sequence. Another glycopeptide unit was also found to be present, in lower yields in both variants 1 (1E) and 2 (2E). The structure of this unit was: Sia alpha 2 yields 3Gal beta1 yields 3GalNAc-Thr. : formula: (see text), alpha 2,6 Sia. Again Thr 345 was the glycosylated amino acid. The amino acid sequence around the glycosylated threonine was found to be NH2-Ala.Pro.Thr(CHO).Ala.Pro.Pro.Glu.

Glycoprotein nature of energy-transducing ATPases: Chemical characterization of glycopeptides isolated from bacterial and chloroplast coupling factors

Glycoprotein nature of energy-transducing ATPases: Chemical characterization of glycopeptides isolated from bacterial and chloroplast coupling factors

Intestinal basement membrane of Ascaris suum. Characterization of carbohydrate units.

Characterization of glycopeptides obtained on alkaline hydrolysis and on extensive collagenase and pronase digestion of the intestinal basement membrane showed the existence of two distinctly different carbohydrate units. One of these is a disaccharide, composed of glucose and galactose, linked to hydroxylysine. It was shown to be identical to the unit (2-O-D-glucopyranosyl-O-D-galactopyranosylhydroxylyasine) present in vertebrate basement membranes, as determined from stability to alkaline hydrolysis, retention time on amino acid analyzer, chemical composition, graded acid hydrolysis, methylation analysis, and periodate oxidation. Direct quantitation after alkaline hydrolysis showed the presence of 9.71 disaccharide units/1000 amino acid residues, indicating that 89% of the hydroxylysine residues of the intestinal membrane are glycosylated. The other unit, consisting of the remaining monosaccharides of the membrane, was separated from the disaccharide unit by gel filtration and ion exchange chromatography of collagenase/pronase digests. Chemical analyses and molecular weight determination by thin layer gel filtration chromatography of purified glycopeptides indicated that this unit is an oligosaccharide which is composed of fucose, galactose, mannose, galactosamine, and glucosamine in a mole ratio of 1:1:1:1:2, respectively. The amount of this unit was calculated to be 2.6 units/1000 amino acid residues.

The Isolation and Characterization of Glycopeptides and Mucopolysaccharides from Plasma Membranes of an Ascites Hepatoma, AH 130 FN1

Plasma membranes were isolated from an ascites hepatoma, AH 130 FN, a free-cell type subline of AH 130, by the fluorescein mercuric acetate (FMA) method. Glycopeptides and mucopolysaccharides were prepared from the membranes by pronase digestion then fractionated chromatographically and electrophoretically. Isolated fractions were analyzed for amino acid and carbohydrate compositions. The results were compared with those for corresponding fractions from AH 66 and AH 130 ((1974) J. Biochem. 76, 319-333; (1975) ibid., 78, 863-872). The fraction excluded from Sephadex G-50 contained mucopolysaccharides and a series of glycopeptides. The mucopolysaccharides were identified as chondroitin sulfate A on the basis of their chemical composition, electrophoretic behavior on cellulose acetate and digestibility with chondroitinase AC [EC 4.2.2.5]. This contrasts with previous findings that mucopolysaccharides from the corresponding fractions from AH 130 and AH 66 were heparan sulfate. The chemical composition of the glycopeptides, which showed high contents of threonine, serine, galactose, galactosamine, glucosamine, and sialic acid, indicated the presence of glycopeptides with O-glycosidic linkages. The glycopeptides also contained a small but significant amount of aspartic acid, suggesting that N-glycosidic glycopeptides were also contained in this fraction. The fraction included in Sepnadex G-50 contaoned N-glycosidic glycopeptides as major components, since the carbohydrate moieties were composed of fucose, galactose, mannose, glucosamine, sialic acid, and a smaller amount of galactosamine. The presence of galactosamine suggested that O-glycosidic glycopeptides were present as minor components. Glycopeptides with both O- and N-glycosidic linkages were isolated from AH 130, but not from AH 66.