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 7–8 h. By using this system, fast and efficient analysis of human serum sialylated N-glycoproteome was achieved. From only 1 μL of human serum, 217 unique glycopeptides and 194 glycosylation sites were successfully identified.
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