Structural fingerprinting of Asn-linked carbohydrates from specific attachment sites in glycoproteins by mass spectrometry: application to tissue plasminogen activator

A sensitive and specific strategy has been developed for determining the sites of attachment of Asn-linked carbohydrates in glycoproteins, and defining the compositions and molecular heterogeneity of carbohydrates at each specific attachment site. In this carbohydrate 'fingerprinting' strategy, potential glycopeptides are identified by comparing the high pressure liquid chromatography (HPLC) chromatograms of proteolytic digests of a glycoprotein obtained before and after digestion with a glycosidase, usually peptide:N-glycosidase F (PNGase F). The glycopeptide-containing HPLC fractions are analyzed by fast atom bombardment mass spectrometry (FAB MS) prior to and after digestion with PNGase F to identify the former glycosylation site peptide and its sequence location (Carr and Roberts, (1986) Anal. Biochem. 157, 396-406). Carbohydrates are extracted from these fractions as the peracetates which are then permethylated and analyzed by FAB MS. The spectra exhibit molecular weight-related ions for each of the parent oligosaccharides present in the fraction which provide composition in terms of hexose, deoxyhexose, N-acetylhexosamine and sialic acid. The relative ratios of these peaks reflect the relative abundances of the various carbohydrate homologs present in the mixture. The derivatives formed are directly amenable to methylation analysis for determination of linkage. This strategy enables the structural classes of carbohydrates at specific attachment sites to be determined using only a few nmol of glycoprotein. The carbohydrate fingerprinting strategy has been applied to a number of glycoproteins including tissue plasminogen activator, the results for which are described herein.     

Analysis of gangliosides using fast atom bombardment mass spectrometry

The native gangliosides GM3, GM1, Fuc-GM1, GD1a, GD1b, Fuc-GD1b, GT1b and GQ1b were analysed by fast atom bombardment mass spectrometry (FAB-MS) in the negative ion mode in a matrix of thioglycerol. After permethylation the same gangliosides were analysed by electron impact (EI) and FAB-MS in the positive ion mode. The negative ion mass spectra furnished information on the molecular weight, the ceramide moiety and the sequence of carbohydrate residues. The sites of attachment and the number of sialic acids present could be deduced directly from the pattern of sequence ions. After addition of sodium acetate positive ion FAB-spectra of the permethylated samples show intense pseudomolecular ions M + Na, that provide evidence on the homogeneity of the samples. In addition, the ceramide part, the oligosaccharide moiety obtained after cleavage of the glycosidic bond of the hexosamine residue, the whole carbohydrate chain and the sialic acids are represented by specific fragment ions. With EI-MS further information can be obtained on the sphingosine and fatty acid components of the ceramide residue. The data show, that the combination of soft ionization mass spectrometry with classical EI-MS gives valuable information on the structure and homogeneity of gangliosides. The method is also applicable to the structural elucidation or quantitation of more complex gangliosides or glycolipid mixtures using only micrograms of material.     

Microanalysis of N-linked oligosaccharides in a glycoprotein by capillary liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

We have studied rapid and simple sugar mapping using liquid chromatography/electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column. The oligosaccharide mixture was separated on the basis of the sequence, branching structure, and linkage, and each oligosaccharide was characterized based on its molecular mass. In this study we demonstrated the usefulness of capillary LC/MS (CapLC/MS) and capillary liquid chromatography/tandem mass spectrometry (CapLC/MS/MS) as sensitive means for accomplishing the structural analysis of oligosaccharides in a low-abundance glycoprotein. The carbohydrate heterogeneity and molecular mass information of each oligosaccharide can be readily obtained from CapLC/MS of a small amount of glycoprotein. CapLC/MS/MS provided b-ion series, which is informative with regard to monosaccharide sequence. Exoglycosidase digestion followed by CapLC/MS elucidated a carbohydrate residue linkage. Using this method, we characterized N-linked oligosaccharides in hepatocyte growth factor produced in mouse myeloma NS0 cells as the complex-type bi-, tri-, and tetraantennary terminated with N-glycolylneuraminic acids and alpha-linked galactose residues. Sugar mapping with CapLC/MS and CapLC/MS/MS is useful for monitoring glycosylation patterns and for structural analysis of carbohydrates in a low-abundance glycoprotein and thus will become a powerful tool in biological, pharmaceutical, and clinical studies.     

The application of mass spectrometry in the structural elucidation of glycosphingolipids

Mass spectrometry has been successfully applied to the analysis of permethylated glycosphingolipids, with and without reduction, as well as of permethylated gangliosides after reduction and silylation. The results obtained by several groups of workers are reviewed. From the data available it can be stated that, with the aid of mass spectrometry, conclusive evidence may be obtained concerning the carbohydrate sequence as far as the typo of sugar is concerned such as hexose, deoxyhexose, hexosamine, and neuraminic acid residues. Branching points can be recognized, and specific fragmentation products allow the differentiation between 1,3 and 1,4-substituted hexosamines of blood-group-active glycosphingolipids (type 1 and type 2 chains). The ceramide residue is documented by several characteristic ions that allow determination not only of sphingosine bases and fatty acid components but also of individual ceramide molecular species. Valuable structural information can thus be obtained to a certain extent also for mixtures of glycosphingolipids composed of different carbohydrate chains or different ceramide residues.     

Protein and carbohydrate structural analysis of a recombinant soluble CD4 receptor by mass spectrometry

The primary structure of a soluble form of the CD4 receptor (sCD4) expressed in Chinese hamster ovary cells has been confirmed by mass spectrometric peptide mapping and and tandem mass spectrometry. These studies corroborated 95% of the 369-amino acid-long sequence and established the fidelity of translation of the NH2 and COOH terminal including the absence of "ragged ends." The arrangement of the three disulfide bonds in recombinant sCD4 was also established by mass spectrometry and comparative high performance liquid chromatography mapping and shown to be identical to that expected from previous studies of intrachain disulfide bonding in T4 antigens derived from sheep and mouse. No other arrangements of disulfides were detected. Carbohydrate mapping by mass spectrometry was used to establish that both potential Asn-linked glycosylation sites in sCD4 (Asn271 and Asn300) have oligosaccharides attached. Structural characterization by mass spectrometry and methylation analysis of the heterogeneous family of oligosaccharides at each of the specific attachment sites indicates that the major components of both families of oligosaccharides have the following biantennary structures: (Formula, see text) where m + n = 0-2, and x = 0,1. Minor carbohydrate components having three N-acetylneuraminic acid (NeuAc) groups and an additional hexose-hexosamine unit were detected by high performance anion-exchange chromatography.     

Purification and structures of branched blood-group-B-active glycosphingolipids from human erythrocyte membranes

Three different variants of complex, branched, highly blood-group-B-active glycosphingolipids (B-III, B-IV, and B-V) have been isolated from human erythrocytes by means of partition of their membranes in n-butanol/phosphate buffer, subsequent removal of nonpolar lipids and proteins by several steps of phase distribution, acetone or sodium acetate precipitation, peracetylation and repeated fractionation of all crude extracts by silicic acid and ion exchange column chromatography. Finally, peracetylated B-glycolipid fractions were purified to homogeneity by preparative silica gel high-performance thin-layer chromatography. Their structures were elucidated by gas chromatographical sugar analysis, by combined gas chromatography/mass spectrometry of partially methylated alditol acetates for the identification of glycosidic linkages, and by fast atom bombardment and electron impact mass spectrometry of the undegraded, permethylated substances in order to establish the molecular mass, sugar sequence, type of oligosaccharide chain, position of hexosyl branching points, number of N-acetyllatosamine units, as well as sphingosine and fatty acid patterns of the ceramide residues. 360-MHz 1H nuclear magnetic resonance spectroscopy in (2H)dimethylsulfoxide of deuterium-exchanged native B-III and B-IV identified all carbohydrate components, their sites of attachment, the anomeric nature of their glycosidic linkages and the sequential arrangement within the oligosaccharide chain. Furthermore, it established the nature of branching points within the carbohydrate sequence, and assigned the different typical saccharide branches to either the position 2 versus 3, or position 3 versus 6 of the 2,3-disubstituted or 3,6-disubstituted galactoses. The nature of the anomeric linkages and branching points of B-V was based upon the series of NMR data obtained from the B-I--B-IV analogues. All results thus establish the following structures: (formula; see text)     

Cell-associated glucans of Burkholderia solanacearum and Xanthomonas campestris pv. citri: a new family of periplasmic glucans.

The cell-associated glucans produced by Burkholderia solanacearum and Xanthomonas campestris pv. citri were isolated by trichloroacetic acid treatment and gel permeation chromatography. The compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry, and high-performance anion-exchange chromatography. B. solanacearum synthesizes only a neutral cyclic glucan containing 13 glucose residues, and X. campestris pv. citri synthesizes a neutral cyclic glucan containing 16 glucose residues. The two glucans were further purified by high-performance anion-exchange chromatography. Methylation analysis revealed that these glucans are linked by 1,2-glycosidic bonds and one 1,6-glycosidic bond. Our 600-MHz homonuclear and 1H-13C heteronuclear nuclear magnetic resonance experiments revealed the presence of a single alpha-1,6-glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The presence of this single alpha-1,6 linkage, however, induces such structural constraints in these cyclic glucans that all individual glucose residues could be distinguished. The different anomeric proton signals allowed complete sequence-specific assignment of both glucans. The structural characteristics of these glucans contrast with those of the previously described osmoregulated periplasmic glucans.     

Fucose containing oligosaccharides from human milk: I. Separation and identification of new constituents

Neutral oligosaccharides isolated from pooled human milk were subjected to fractionation on high-performance thin-layer chromatography (HPTLC) plates, Iatrobeads, and reverse-phase chromatography after borohydride reduction and peracetylation. By the combined HPLC and HPTLC separation a mixture of pooled human milk oligosaccharides was separated into 101 fractions. These fractions were characterized by field desorption or fast atom bombardment (FAB)-mass spectrometry. Each of the carbohydrate constituents, the peracetylated glucitol, the galactose, the glucosamine, and the fucose contribute specific mass increments to the molecular weight of the oligosaccharide. Therefore, the exact carbohydrate composition can be calculated from the molecular weight determined by mass spectrometry. Among the fractions obtained one trifucosyl-lacto-N-tetraose, five monofucosyl-, eleven difucosyl-, and nine trifucosyl-lacto-N-hexaoses, one monofucosyl-, eight difucosyl-, seven trifucosyl-, four tetrafucosyl-, and two pentafucosyl-lacto-N-octaoses, one trifucosyl-, and two difucosyl-lacto-N-decaoses could be identified. FAB spectra furnished additional data on structural features of the isolated oligosaccharides.     

Structural characterization of glycosphingolipids by direct chemical ionization mass spectrometry

This report describes the use of direct chemical ionization mass spectrometry with ammonia as the reagent gas (NH3-DCI) for structure analysis of underivatized, permethylated and permethylated and reduced glycosphingolipids. In contrast to ionization by electron impact, the NH3-DCI mass spectra exhibit intense molecular and carbohydrate sequence-related ions using microgram amounts of sample. Underivatized glycosphingolipids with up to two sugar residues yield abundant protonated and ammonia-cationized molecular ions and structurally significant fragments. Permethylation in conjunction with NH3-DCI can be used to obtain molecular weight as well as oligosaccharide sequence and branching information on neutral, acidic and complex-type glycosphingolipids with up to five sugar residues. Reduction of the permethylated derivatives gives rise to several new, structurally significant fragments in the corresponding NH3-DCI mass spectra which enable fatty acid and base compositions to be determined. Isotopically labeled reagent gases have been used to confirm the assignment of fragment structures and to demonstrate that the ions observed are unique to the NH3-DCI mass spectra.     

Assignment of anomeric configurations of pyranose sugars in oligosaccharides using a sensitive FAB-MS strategy

This paper describes a sensitive strategy employing fast atom bombardment mass spectrometry (FAB-MS) for defining the anomeric configurations of pyranose sugars in oligosaccharides. The method, which is applicable to mixtures of reduced or unreduced oligosaccharides, is based upon FAB-MS analyses of deuteroacetylated derivatives before and after oxidation with chromium trioxide. This reagent, whose potential value in carbohydrate chemistry was first recognized by Angyal and which was subsequently more fully exploited by Lindberg, oxidizes beta-pyranosides to keto-esters leaving alpha-pyranosides largely intact. In this paper we show that the products of chromium trioxide oxidation can be successfully analysed at the microgram level using FAB-MS. The molecular and fragment ions produced in the FAB experiment define the number of sites oxidized and their location in the sequence. For samples which fragment poorly we describe a mild methanolysis procedure, compatible with FAB-MS, which preferentially cleaves the esters formed during the oxidation. Incorporation of an acetolysis step prior to oxidation permits analyses of polysaccharides. This oxidation/FAB-MS strategy should prove valuable in structural analyses of a wide range of biologically important carbohydrates which cannot be isolated in sufficient quantities to permit nuclear magnetic resonance studies.     

Analysis of protein glycosylation by mass spectrometry

There is a growing pharmaceutical market for protein-based drugs for use in therapy and diagnosis. The rapid developments in molecular and cell biology have resulted in production of expression systems for manufacturing of recombinant proteins and monoclonal antibodies. These proteins are glycosylated when expressed in cell systems with glycosylation ability. For glycoproteins intended for therapeutic administration it is important to have knowledge about the structure of the carbohydrate side chains to avoid cell systems that produce structures, which in humans can cause undesired reactions, e.g., immunological and unfavorable serum clearance rate. Structural analysis of glycoprotein oligosaccharides requires sophisticated instruments like mass spectrometers and nuclear magnetic resonance spectrometers. However, before the structural analysis can be conducted, the carbohydrate chains have to be released from the protein and purified to homogeneity, and this is often the most time-consuming step. Mass spectrometry has played and still plays an important role in analysis of protein glycosylation. The superior sensitivity compared to other spectroscopic methods is its main asset. Structural analysis of carbohydrates faces several problems, however, due to the chemical nature of the constituent monosaccharide residues. For oligosaccharides or glycoconjugates, the structural information from mass spectrometry is essentially limited to monosaccharide sequence, molecular weight, and only in exceptional cases glycosidic linkage positions can be obtained. In order to completely establish an oligosaccharide structure, several other structural parameters have to be determined, e.g., linkage positions, anomeric configuration and identification of the monosaccharide building blocks. One way to address some of these problems is to work on chemical pretreatment of the glycoconjugate, to specifically modify the carbohydrate chain. In order to introduce specific modifications, we have used periodate oxidation and trifluoroacetolysis with the objective of determining glycosidic linkage positions by mass spectrometry.     

Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast.

The N-glycans of purified recombinant middle surface protein (preS2+S) from hepatitis B virus, a candidate vaccine antigen expressed in a mnn9 mutant strain of Saccharomyces cerevisiae, have been characterized structurally. The glycans were released by N-glycanase treatment, isolated by size-exclusion chromatography on Sephadex G-50 and Bio-Gel P-4 columns, and analyzed by 500-MHz 1H NMR spectroscopy and fast atom bombardment mass spectrometry. The mixture of oligosaccharides was fractionated by HPLC, the major subfractions were isolated, and their carbohydrate compositions were determined by high-pH anion-exchange chromatography with pulsed amperometric detection. The combined results suggest that high-mannose oligosaccharides account for all the N-glycans released from preS2+S: structures include Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 isomers in the ratios of 3:6:1. Approximately 80% of the oligosaccharides contain the C2,C6-branched trimannosyl structural element typical of yeast high-mannose oligosaccharides but not usually found in high-mannose oligosaccharides in animal glycoproteins.     

The linkage between O-specific caryan and core region in the lipopolysaccharide of Burkholderia caryophylli is furnished by a primer monosaccharide

From the lipopolysaccharide (LPS) fraction of the plant-pathogenic bacterium Burkholderia caryophylli, the linkage between O-specific caryan and core region was characterised. The LPS fraction was first treated with 48% aqueous HF at 4 degrees C and successively with 1% acetic acid at 100 degrees C. A main oligosaccharide representing the carbohydrate backbone of the core region and a portion of the caryan (three unit of caryose) was isolated by high-performance anion-exchange chromatography. Compositional and methylation analyses, matrix-assisted laser desorption/ionisation mass spectrometry and 2D NMR spectroscopy identified the structure: [carbohydrate structure: see text]. The above residues are alpha-linked pyranose rings, if not stated otherwise. Hep is L-glycero-D-manno-heptose, Car is 4,8-cyclo-3,9-dideoxy-L-erythro-D-ido-nonose and Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid. This finding indicates that QuiNAc residue is the primer monosaccharide, which connects the core oligosaccharide to caryan O-chain.     

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.     

Characterization of glycosphingolipid mixtures with up to ten sugars by gas chromatography and gas chromatography-mass spectrometry as permethylated oligosaccharides and ceramides released by ceramide glycanase

A novel, effective method for structural characterization of glycosphingolipids has been devised. It employs ceramide glycanase to release intact oligosaccharides followed by analysis using high-mass gas chromatography-mass spectrometry. The oligosaccharides and ceramides released by the glycanase were permethylated and analyzed. The capillary gas chromatography gave excellent resolution and separated, for example, two isomeric 10-sugar oligosaccharides with a molecular mass of 2150 daltons differing only by a Gal1-3GlcNAc and a Gal1-4GlcNAc linkage. The oligosaccharides released from sialic acid containing glycosphingolipids (gangliosides) were also analyzed for monosialo compounds. This analytical approach is simple, is quick, and can readily allow quantitation of individual glycosphingolipids.     

A general and sensitive chemical method for sequencing the glycosyl residues of complex carbohydrates

This paper describes a new glycosyl-sequencing method. This method was made possible by the ability to fractionate complex mixtures of peralkylated oligosaccharides by reversed-phase, high-pressure liquid chromatography. The fractionation ability of the reversed-phase system allows the isolation and subsequent unambiguous identification by g.l.c.-m.s. of disaccharides, almost all trisaccharides, and, in some cases, tetrasaccharides generated by successive partial acid hydrolysis, reduction, and ethylation of a permethylated, complex carbohydrate. As these small oligosaccharides overlap within the unhydrolyzed, complex carbohydrate, the oligosaccharide sequences may be pieced together, and, with the glycosyl-linkage composition of the intact complex carbohydrate, can be used to determine the glycosyl sequence of the complex carbohydrate. The details of the sequencing method are illustrated by the elucidation of the glycosyl sequences of three complex carbohydrates. These examples demonstrate the wide variety of complex carbohydrates whose structures can be ascertained by the new sequencing technique. Two of the examples are the commercially available polysaccharides, lichenan and xanthan, whose structures have already been reported. The other example is a nonasaccharide derived from xyloglucan, a structural polymer of plant cell-walls. The glycosyl residues of the complex carbohydrates studied include hexosyl, deoxyhexosyl, pentosyl, glycosyluronic, and pyruvic acetal-substituted hexosyl residues. It will be demonstrated that the new glycosyl-sequencing technique is not compromise by the presence, in the carbohydrate to be analyzed, of glycosyl linkages possessing very different acid labilities. Two major advantages of this sequencing technique are that it is relatively rapid and that it requires only milligram quantities of carbohydrate.     

Rapid characterization of mucin oligosaccharides from rat small intestine with gas chromatography-mass spectrometry

Mucin glycopeptides were isolated from rat small intestinal mucosa after reduction/alkylation, trypsin digestion and gel chromatography. The oligosaccharides were released by using alkaline-NaBH4, separated into neutral and acidic species and permethylated. The derivatized mixtures were analysed with fast atom bombardment mass spectrometry and gas chromatography-mass spectrometry using thin film columns. Permethylated neutral oligosaccharides with up to seven sugars could be chromatographed and detected with mass spectrometry. The complex mixture revealed was partly due to the linkage GalNAc being substituted at both position 3 and 6. The approach will be very useful when analysing small amounts of mucins and mucin fragments.     

Osmoregulated periplasmic glucans of the free-living photosynthetic bacterium Rhodobacter sphaeroides.

The osmoregulated periplasmic glucans (OPGs) produced by Rhodobacter sphaeroides, a free-living organism, were isolated by trichloracetic acid treatment and gel permeation chromatography. Compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry and nuclear magnetic resonance. R. sphaeroides predominantly synthesizes a cyclic glucan containing 18 glucose residues that can be substituted by one to seven succinyl esters residues at the C6 position of some of the glucose residues, and by one or two acetyl residues. The glucans were subjected to a mild alkaline treatment in order to remove the succinyl and acetyl substituents, analyzed by MALDI mass spectrometry and purified by high-performance anion-exchange chromatography. Methylation analysis revealed that this glucan is linked by 17 1,2 glycosidic bonds and one 1,6 glycosidic bond. Homonuclear and (1)H/(13)C heteronuclear NMR experiments revealed the presence of a single alpha-1,6 glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The different anomeric proton signals allowed a complete sequence-specific assignment of the glucan. The structural characteristics of this glucan are very similar to the previously described OPGs of Ralstonia solanacearum and Xanthomonas campestris, except for its different size and the presence of substituents. Therefore, similar OPGs are synthesized by phytopathogenic as well as free-living bacteria, suggesting these compounds are intrinsic components of the Gram-negative bacterial envelope.     

N-linked oligosaccharide structures in the diamine oxidase from porcine kidney

Structures of the N-linked glycans released from porcine kidney diamine oxidase (DAO) were characterized utilizing various analytical techniques, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), high-performance capillary electrophoresis (HPCE), and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The oligosaccharide sequences present in DAO were conclusively determined using specific exoglycosidases in conjunction with MALDI/TOF-MS. The structures found in the glycoprotein are primarily linear, di-, or tribranched fucosylated complex type. MS analysis of the esterified N-glycan pool derived from DAO indicated the presence of several di- and trisialylated structures.