TY - JOUR
T1 - Site-specific characterization of the N-linked oligosaccharides of a murine immunoglobulin M by high-performance liquid chromatography/electrospray mass spectrometry
AU - Wang, Fang
AU - Nakouzi, Antonio
AU - Angeletti, Ruth Hogue
AU - Casadevall, Arturo
N1 - Funding Information:
The National Institutes of Health (NIH Grants AI33774, AI3342, and HL-59842-01 to A.C.) is acknowledged for financial support. The authors also gratefully acknowledge Drs. Olga O. Blumenfeld, Pamela Stanley, and Daniel Moloney for critical reading the manuscript and helpful discussion. F.W. thanks Edward Nieves helpful discussion in experiments.
PY - 2003/3/15
Y1 - 2003/3/15
N2 - 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.
AB - 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.
KW - Glycoprotein
KW - Glycosylation
KW - HPLC/ESI-MS/MS
KW - HPLC/ESI-TOF MS
KW - Monoclonal immunoglobulin M
KW - N-linked oligosaccharides
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U2 - 10.1016/S0003-2697(02)00693-0
DO - 10.1016/S0003-2697(02)00693-0
M3 - Article
C2 - 12654314
AN - SCOPUS:12244254420
SN - 0003-2697
VL - 314
SP - 266
EP - 280
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -