Journal
GLYCOCONJUGATE JOURNAL
Volume 29, Issue 5-6, Pages 433-443Publisher
SPRINGER
DOI: 10.1007/s10719-012-9404-3
Keywords
Sialoglycopeptides; N-glycosylation site; Peptide IPG-IEF; Lectin; TiO2 chromatography; LC-ESI-MS/MS
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Funding
- National 973/S973/863 and NSF projects [S973-2011CB910600, NFS-31100590, 20975024, S973-2010CB912700, 863-2012AA020200]
- Shanghai Municipal Natural Science Foundation [11ZR1403000]
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Playing an important role in a broad range of biological and pathological processes, sialylation has been drawing wide interest. The efficient sialoglycopeptides enrichment methods are therefore attracting considerable attention. In this paper, we first compared two conventional enrichment methods, lectin and TiO2, and analyzed their characteristics. Furthermore, considering the highly negatively charged nature of sialic acids, we developed a new strategy, peptide immobilized pH gradient isoelectric focusing (IPG-IEF) assisted TiO2 chromatography (PIAT), for the highly efficient enrichment of sialoglycopeptides. In this method, peptides were first separated into 24 fractions using peptide IPG-IEF. Sialoglycopeptides were relatively concentrated in low-pH fractions of the immobilized pH strips and were captured using TiO2 chromatography. As a result, 614 N-glycosylation sites were identified in 582 sialoglycopeptides within 322 sialoglycoproteins from rat liver using PIAT. To our knowledge, this work represents one of the most comprehensive sialoglycoproteomic analyses in general and exhibits the largest database of sialoglycoproteome in rat liver currently. So the new strategy introduced here exhibits high efficiency and universality in the sialoglycopeptide enrichment, and is a powerful tool for sialoglycoproteome exploration.
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