Site-specific characterization of the N-linked oligosaccharides of a murine immunoglobulin M by high-performance liquid chromatography/electrospray mass spectrometry

Fang Wang, Antonio Nakouzi, Ruth Hogue Angeletti, Arturo Casadevall

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Immunoglobulin M is an especially important product of the immune system because it plays a critical role in early protection against infections. In this report, the glycosylation pattern of the protective murine monoclonal IgM 12A1 to Cryptococcus neoformans polysaccharide was analyzed by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Peptide mapping studies covering 88% of the deduced amino acid sequence indicated that of the six potential N-glycosylation sites in this antibody only five were utilized, as the tryptic peptide derived from monoclonal IgM 12A1 containing Asn-260 was recovered without carbohydrates. The oligosaccharide side chains of monoclonal IgM 12A1 were characterized at each of the N-glycosylation sites. Asn-166 possessed 20 monosialylated and nonsialylated, and fucosylated and nonfucosylated complex- and hybrid-type oligosaccharides and one high-mannose-type oligosaccharide. Thirteen oligosaccharides were attached to the site at Asn-401, including six complex-type, four hybrid-type, and three high-mannose-type oligosaccharides. Twelve hybrid-type oligosaccharides were attached to Asn-378, three of which had terminal sialic acids. Eleven hybrid-type oligosaccharides were attached to Asn-331, seven of which had terminal sialic acids. Only two high-mannose type oligosaccharides were attached to Asn-363. These results indicated great complexity in the structure and composition of oligosaccharides attached to individual IgM glycosylation sites.

Original languageEnglish (US)
Pages (from-to)266-280
Number of pages15
JournalAnalytical Biochemistry
Volume314
Issue number2
DOIs
StatePublished - Mar 15 2003

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High performance liquid chromatography
Oligosaccharides
Mass spectrometry
Immunoglobulin M
Mass Spectrometry
High Pressure Liquid Chromatography
Glycosylation
Mannose
Sialic Acids
Electrospray ionization
Peptides
Cryptococcus neoformans
Peptide Mapping
Electrospray Ionization Mass Spectrometry
Immune system
Polysaccharides
Amino Acid Sequence
Immune System
Carbohydrates
Amino Acids

Keywords

  • Glycoprotein
  • Glycosylation
  • HPLC/ESI-MS/MS
  • HPLC/ESI-TOF MS
  • Monoclonal immunoglobulin M
  • N-linked oligosaccharides

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Site-specific characterization of the N-linked oligosaccharides of a murine immunoglobulin M by high-performance liquid chromatography/electrospray mass spectrometry. / Wang, Fang; Nakouzi, Antonio; Angeletti, Ruth Hogue; Casadevall, Arturo.

In: Analytical Biochemistry, Vol. 314, No. 2, 15.03.2003, p. 266-280.

Research output: Contribution to journalArticle

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