Assessment of glycosylation-site heterogeneity using plasma desorption mass spectrometry

R. Reid Townsend, Mehrshid Alai, Mark R. Hardy, Catherine C. Fenselau

Research output: Contribution to journalArticle

Abstract

Plasma desorption mass spectrometry (PD-MS) was used to assess the molecular weight heterogeneity of glycopeptides (6-12 amino acids) from each of the three N-linked glycosylation sites of bovine fetuin (R. G. Spiro (1962) J. Biol. Chem. 237, 382-388). The glycopeptides were purified by a combination of anion exchange chromatography and reverse-phase HPLC. Since no detectable fragmentation was observed in the PD-MS of these asialoglycopeptides, the observation of multiple molecular ions could be attributed to either carbohydrate or peptide heterogeneity. Assignment of molecular ions, within 3 to 5 amu of the theoretical mass, of glycopeptides from each glycosylation site was made from amino acid composition, peptide sequence around the glycosylation sites, and previously reported triantennary oligosaccharide structures (B. Nilsson, N. E. Nordén, and S. Svensson (1979) J. Biol. Chem. 254, 4545-4553). Ion groups differing in mass by one N-acetyllactosamine unit were observed in glycopeptides from the Asn-Asp and Asn-Cys sites, localizing these previously observed biantennary oligosaccharide structures (R. R. Townsend, M. R. Hardy, T. C. Wong, and Y. C. Lee (1986) Biochemistry 25, 5716-5725; S. Takasaki and A. Kobata (1986) Biochemistry 25, 5709-5715) to these two sites. The presence of biantennary oligosaccharides at the Asn-Asp sites could be substantiated using 1H NMR but were not detected in the Asn-Cys glycopeptides. PD-MS was also implemented in the purification protocol for these glycopeptides and proved to be useful in assessing purity of chromatographic fractions which were mixtures of glycopeptides displaying both carbohydrate and peptide heterogeneity. A preparation scheme was developed to obtain molecular ions of desialylated glycopeptides by PD-MS.

Original languageEnglish (US)
Pages (from-to)180-191
Number of pages12
JournalAnalytical Biochemistry
Volume171
Issue number1
DOIs
StatePublished - May 15 1988

Keywords

  • carbohydrates
  • fetuin
  • glycoproteins
  • HPLC
  • mass spectrometry
  • NMR
  • peptides

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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