Andrew J. Reason

Mass spectrometry of carbohydrate-containing biopolymers

Publisher Summary This chapter discusses practical aspects of fast atom bombardment–mass spectrometry (FAB–MS) and electrospray–mass spectrometry (ES–MS), including data interpretation, and describes examples of experimental strategies incorporating all three mass spectrometric methods, which are best suited to the analysis of glycoproteins, complex carbohydrates (including glycolipids), and glycosaminoglycans, respectively. In ES–MS technique a stream of liquid containing the sample of interest is injected directly into the ES source where the sample molecules are stripped of solvent, leaving them as multiply charged species whose charges reflect the number of functional groups that can be protonated (positive ion mode) or deprotonated (negative ion mode) at the pH of the carrier liquid. Two fragmentation pathways dominate carbohydrate FAB-MS, namely, A-type cleavage and β cleavage. A-type cleavage occurs on the nonreducing side of glycosidic bonds to give oxonium-type fragment ions and is the dominant pathway for permethyl and peracetyl derivatives. Cleavage is favored at amino sugar residues. A-type fragment ions are extremely helpful for structure assignments. β cleavage also involves glycosidic fission but, because a hydrogen transfer accompanies bond breakage, no charge is produced at the actual cleavage site as occurs during A-type cleavage.

Identification of Major Outer Surface Proteins of Streptococcus agalactiae

ABSTRACT To identify the major outer surface proteins of Streptococcus agalactiae (group B streptococcus), a proteomic analysis was undertaken. An extract of the outer surface proteins was separated by two-dimensional electrophoresis. The visualized spots were identified through a combination of peptide sequencing and reverse genetic methodologies. Of the 30 major spots identified as S. agalactiae specific, 27 have been identified. Six of these proteins, previously unidentified in S. agalactiae, were sequenced and cloned. These were ornithine carbamoyltransferase, phosphoglycerate kinase, nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, purine nucleoside phosphorylase, enolase, and glucose-6-phosphate isomerase. Using a gram-positive expression system, we have overexpressed two of these proteins in an in vitro system. These recombinant, purified proteins were used to raise antisera. The identification of these proteins as residing on the outer surface was confirmed by the ability of the antisera to react against whole, live bacteria. Further, in a neonatal-animal model system, we demonstrate that some of these sera are protective against lethal doses of bacteria. These studies demonstrate the successful application of proteomics as a technique for identifying vaccine candidates.

Structural analysis of laminarans by MALDI and FAB mass spectrometry

Abstract MALDI and FAB mass spectrometry were applied to eight samples of laminarans from different algal species. The existence of both M-chains (mannitol-containing) and G-chains (mannitol-free) was confirmed for six of them, as well as the absence of M-chains for laminarans from two Cystoseira sp. It was found that Cystoseira barbata and C. crinita glucans contain a small percentage of N-acetylhexosamine-terminated chains. This is the first observation of nitrogen-containing sugars in laminarans. The presence of cyclic structures in some laminaran samples is suggested by the MALDI and FAB data. This study demonstrates the power of modern mass spectrometry for defining d.p. profiles in complex mixtures of polysaccharides and for rapidly and sensitively revealing the presence of novel components.

Carbohydrate moieties in human secretory component.

Human secretory component has seven putative sites for N-linked glycosylation. From tryptic and Glu-C digests we have isolated peptides encompassing asparagines 65, 72, 117, 168, 403, 451 and 481. Analysis by on line HPLC-electrospray mass spectrometry indicated that these residues were fully glycosylated and that the major carbohydrate moieties were far less diversified in composition than expected. Fast atom bombardment mass spectrometry performed on oligosaccharides released by peptide-N-glycosidase F treatment of fractionated and unfractionated SC digests showed the following glycan compositions: Fuc(2)Hex(5)HexNAc(4), Fuc(3)Hex(5)HexNAc(4), NeuAcFucHex(5)HexNAc(4), NeuAcFuc(2)Hex(5)HexNAc(4), NeuAc(2)Hex(5)HexNAc4 and NeuAc(2)FucHex(5)HexNAc(4). Three of these oligosaccharides are the major carbohydrate moieties in human lactoferrin. A possible biological role of the secretory component glycans in the protection of mucosal surfaces is discussed.

Human free secretory component is composed of the first 585 amino acid residues of the polymeric immunoglobulin receptor

The main objective of this work was to unequivocally determine the C‐terminal sequence of human milk free secretory component (SC). It was found to end at arginine‐585, i.e. 33 amino acids downstream from the major heterogeneous C‐terminal residue previously identified for colostrum SC. In contrast, our data showed that the C‐terminal end of SC was found to be homogeneous. Conflicting assignments, Asp/Gln, a missing Asn‐211, Asp/Asn, Glu/Gln were corrected and found to agree with the cDNA sequence. An Ala/Val substitution at position 562 (domain VI) was identified. Its genetic significance is uncertain at present.

Structural Characterization of the Inflammatory Moiety of a Variable Major Lipoprotein of Borrelia recurrentis

Louse-borne relapsing fever, caused byBorrelia recurrentis, provides one of the best documented examples of the causative role of tumor necrosis factor (TNF) in the pathology of severe infection in humans. We have identified the principal TNF-inducing factor of B. recurrentis as a variable major lipoprotein (Vmp). Here we report the complete gene sequence of Vmp, including its lipoprotein leader sequence. Using metabolically labeled forms of the native Vmp we confirm that the TNF inducing properties are associated with the lipid portion of the molecule. Quadrupole orthogonal time of flight mass spectrometry unequivocally locates the lipidic moiety at the NH2-terminal cysteine of the native polypeptide, and indicates the existence of three forms which are consistent with the structures C16:0, C16:0, C16:0 glyceryl cysteine; C18:1, C16:0, C16:0 glyceryl cysteine; and C18:0, C16:0, C16:0 glyceryl cysteine. These data provide the first direct evidence that the TNF inducing lipid modification of native Borrelia lipoproteins is a structural homologue of the murein lipoprotein of Escherichia coli.

A partial reductive-cleavage study of the capsular polysaccharide of Escherichia coli K57

Trideuteriomethylated and methylated derivatives of the capsular polysaccharide of Escherichia coli K57 were partially cleaved by Et3SiH, using Me3SiOSO2 Me and Me3SiOSO2CF3 as catalysts, to produce oligosaccharide-anhydroalditols. The structures of the trideuteriomethylated trisaccharide- and tetrasaccharide-anhydroalditols isolated were established by FABMS and NMR spectroscopy. Although conditions for the selective production of the tetrasaccharide-anhydroalditol could not be established, oligosaccharide-anhydroalditols were isolated in sufficiently high yield to make this an attractive approach for the structural elucidation of the repeating units of bacterial polysaccharides.

Determination of the structure of the capsular antigen of Escherichia coli O8:K46:H30, using FABMS and 2D-NMR spectroscopy

The structure of the capsular antigen from Escherichia coli O8:K46:H30 was elucidated by methylation analysis and 1D and 2D 1H- and 13C-NMR spectroscopy, and by methylation analysis, 1D- and 2D-NMR spectroscopy, and FABMS of the oligosaccharide-alditol obtained after dephosphorylation of the polymer with aqueous hydrofluoric acid. The capsular polymer is of the teichoic acid type and has the following repeating unit. [Formula: see text]

Characterisation of the N-linked oligosaccharides of the light chain of human glycoprotein IIb by f.a.b.-m.s.

The glycosylation of the light chain (GPIIbL) of glycoprotein IIb, one of the glycoproteins constituting the receptor for fibrinogen, fibronectin, and the von Willebrand factor on platelet cell surfaces, was investigated using fast-atom-bombardment mass spectrometry (f.a.b.-m.s.). Complex-type N-glycans were observed, attached to Asn-60. The most abundant oligosaccharide is a disialylated biantennary structure substituted with fucose on the chitobiose core. Mono-sialylated biantennary, and di- and tri-sialylated triantennary structures were found as minor constituents of the N-glycan population. The amino acid sequence of GPIIbL was fully mapped by f.a.b.-m.s., thereby providing the first direct evidence for the absence of O-glycosylation.

S9.11 Mass-spectrometric analysis of the glycosylation of the enzyme lecithin-cholesterol acyl transferase

R. J. Ha rrisl, H. van Halbeek 2, J. Glushka 2, L. J. Basa ~, V. T. Ling ~, K. J. Smith 3 and M. W. Spellman l ~Department of Medicinal and Analytical Chemistry, Genentech, Inc., 460 Pt. San Bruno Blvd., So. San Francisco, CA, USA.; 2Complex Carbohydrate Research Center and Department of Biochemistry, The University of Georgia, Athens, GA, USA.; and 3Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM, USA.