Journal
PROTEOMICS
Volume 13, Issue 8, Pages 1306-1313Publisher
WILEY
DOI: 10.1002/pmic.201200367
Keywords
ACN-improved digestion; Filter-aided sample preparation; Glycoproteomics; Human serum; Sialic acid-containing N-glycopeptide; Ti(IV)-IMAC
Funding
- Creative Research Group Project by NSFC [21021004]
- National Natural Sciences Foundation of China [21105101]
- China State Key Basic Research Program Grant [2012CB910101, 2012CB910601]
- National Key Special Program on Infection diseases [2012ZX10002009-011]
- Analytical-Method Innovation Program of MOST [2010IM030500]
- Facility Promotion Program from Chinese Academy of Sciences [Y2010041]
- Knowledge Innovation program of DICP
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Terminal sialylation is very important in cancer biology and has been extensively investigated for the discovery of potential clinical biomarkers of cancers. In this study, we presented a novel approach, by using of Ti(IV)-IMAC, to enrich sialic-acid-containing N-glycopeptides for the analysis of terminal sialylation. Compared with conventional method using TiO2, this approach obtained 2.5 times more glycopeptides and glycosylation sites. Then, a simple integrated system combining filter-aided sample preparation, ACN-improved digestion, and Ti(IV)-IMAC enrichment was established for efficient analysis. In this system, protein digestion, glycopeptide enrichment, and deglycosylation were integrated and were performed sequentially in a single filter unit without any need for desalting, lyophilization, or sample transfer procedures. As a result, the number of identifications was improved by 1.5-fold and the total processing time was drastically reduced to only 78 h. By using this system, fast and efficient analysis of human serum sialylated N-glycoproteome was achieved. From only 1 L of human serum, 217 unique glycopeptides and 194 glycosylation sites were successfully identified.
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