We describe here a simple and integrated spintip-based glycoproteomics technology,termed Glyco-SISPROT, for challenging the lowest starting material limitation and achieving a comprehensive view of glycoproteome with shortest sample processing time. By carefully integrating and optimizing SCX, C18 and Concanavalin A packing material and their combination in spintip format, both predigested peptides and protein lysates could be processed by Glyco-SISPROT with high efficiency. More importantly, deglycopeptide, intact glycopeptide and glycans released by multiple glycosidases could be readily collected from the same Glyco-SISPROT workflow for LC-MS analysis. In total, above 1850 glycosites in 1770 unique deglycopeptides were characterized from mouse liver by using either 100 macrograms of predigested peptides or directly using 100 macrograms of protein lysates, in which about 30 percentage of glycosites were released by both PNGase F and Endos. To the best of our knowledge, this approach should be one of the most comprehensive glycoproteomic approaches by using limited protein starting material. One significant benefit of Glyco-SISPROT was that whole processing time was dramatically reduced from a few days to only 6 hours with good reproducibility when protein lysates were directly processed by Glyco-SISPROT.
[doi:10.25345/C5JK70]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: glycoproteomics
Principal Investigators: (in alphabetical order) |
Ruijun Tian, SUSTech, China |
Submitting User: | Peiwu_1213 |
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