title: Site-specific characterization of glycoprotein carbohydrates by exoglycosidase digestion and laser desorption mass spectrometry.
abstract: A rapid and sensitive method for sequencing oligosaccharides has been developed, using matrix-assisted laser desorption mass spectrometry to monitor the digestion of glycopeptides by specific exoglycosidases. Recombinant human tissue inhibitor of metalloproteinases (TIMP), which has two glycosylation sites, has been characterized to illustrate this new approach for obtaining site-specific information. Glycopeptides which span residues Asn30 and Asn78 were generated by tryptic digestion of 1 nmol of TIMP and separated by reverse-phase high-performance liquid chromatography. The oligosaccharide composition of the glycoforms was inferred from the observed mass shifts following digestion by peptide-N-glycosidase F. Composition and sequence were then elucidated by digestion with specific exoglycosidases, using a total of 200 pmol of each glycopeptide. Glycopeptides from well-characterized proteins, fetuin, alpha 1-acid glycoprotein, and tissue plasminogen activator were also analyzed to confirm exoglycosidase specificity for glycopeptides and establish the quantitative significance of the relative intensities of peaks in the mass spectra. Both TIMP glycosylation sites exhibited extensive heterogeneity comprising mainly fucosylated complex oligosaccharides, but in different proportions. The Asn78 site also contained 4.4% nonfucosylated mannose (Man4) oligosaccharide. The merits and limitations of this approach as a universal method for oligosaccharide analysis are discussed.
authors.affiliation: Finnigan MAT Ltd., Paradise, Hemel Hempstead, Hertfordshire, United Kingdom.
authors.forename: C W
authors.initials: CW
authors.lastname: Sutton
authors.forename: J A
authors.initials: JA
authors.lastname: O'Neill
authors.forename: J S
authors.initials: JS
authors.lastname: Cottrell
mesh_terms: D000581:Amidohydrolases; D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D006020:Glycopeptides / Q000032:analysis*; D006023:Glycoproteins / Q000032:analysis*; D006026:Glycoside Hydrolases; D007834:Lasers; D013058:Mass Spectrometry / Q000379:methods*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D019714:Tissue Inhibitor of Metalloproteinases
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; D009844:Oligosaccharides; D019714:Tissue Inhibitor of Metalloproteinases; D006026:Glycoside Hydrolases; D008666:Metalloendopeptidases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase