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Mol Cell Proteomics. 2010 Nov;9(11):2354-68. doi: 10.1074/mcp.M110.000430. Epub 2010 Jun 22.

Sialic acid-focused quantitative mouse serum glycoproteomics by multiple reaction monitoring assay.

Author information

1
Graduate School of Life Science, Frontier Research Center for the Post-Genomic Science and Technology, Hokkaido University, Kita-ku, Sapporo, Japan.

Abstract

Despite increasing importance of protein glycosylation, most of the large-scale glycoproteomics have been limited to profiling the sites of N-glycosylation. However, in-depth knowledge of protein glycosylation to uncover functions and their clinical applications requires quantitative glycoproteomics eliciting both peptide and glycan sequences concurrently. Here we describe a novel strategy for the multiplexed quantitative mouse serum glycoproteomics based on a specific chemical ligation, namely, reverse glycoblotting technique, focusing sialic acids and multiple reaction monitoring (MRM). LC-MS/MS analysis of de-glycosylated peptides identified 270 mouse serum peptides (95 glycoproteins) as sialylated glycopeptides, of which 67 glycopeptides were fully characterized by MS/MS analyses in a straightforward manner. We revealed the importance of a fragment ion containing innermost N-acetylglucosamine (GlcNAc) residue as MRM transitions regardless the sequence of the peptides. Versatility of the reverse glycoblotting-assisted MRM assays was demonstrated by quantitative comparison of 25 targeted glycopeptides from 16 proteins between mice with homo and hetero types of diabetes disease model.

PMID:
20571061
PMCID:
PMC2984228
DOI:
10.1074/mcp.M110.000430
[Indexed for MEDLINE]
Free PMC Article

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