期刊
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
卷 1723, 期 1-3, 页码 229-239出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbagen.2005.02.013
关键词
disaccharide; ion trap; multistage MS/MS; plant glycosylation
Horseradish peroxidase (HRP) is widely used in biomedical research as a reporter enzyme in diagnostic assays. In addition, it is of considerable interest as a model glycoprotein with core-xylosylated and -(alpha 1-3)-fucosylated N-glycans that form antigenic elements of plant allergens and parasitic helminths. Using a combination of techniques comprising (1) nano-liquid chromatography (LC)-mass spectrometry (MS)IMS with multiple selection/fragmentation cycles of HRP tryptic (glyco-)peptides, (2) nano-electrospray MS of intact HRP, and (3) carbohydrate linkage analysis, it was revealed that most of the HRP N-glycosylation sites can be occupied with an alternative Fuc(1-3)GlcNAc-disaccharide. Two main variants of HRP occur: The major population (approximately 60%) has eight glycosylation sites carrying core(1-3)fucosylated, xylosylated, trimannosyl N-glycans, with the ninth potential N-glycosylation site Asn(316) not occupied. Another group of HR-P carries seven of the above-mentioned N-glycans, with an eighth N-glycosylation site carrying the alternative Fuc(1-3)GlcNAc-unit (approximately 35%). In addition, minor subsets of HRP were found to contain a xylosylated, trimannosyl N-glycan lacking core-fucosylation as a ninth N-glycan attached to Asn316, which has hitherto been assumed to be unoccupied. The finding of these new features of glycosylation of an already exceptionally well-studied glycoprotein underscores the potential of the nano-LC-MSn based analytical approach followed. (c) 2005 Elsevier B.V. All rights reserved.
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