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An integrated strategy for high-sensitive and multi-level glycoproteome analysis from low micrograms of protein samples
- Gao, Weina, Li, Hongjie, Liu, Liping, Huang, Peiwu, Wang, Zhikun, Chen, Wendong, Ye, Mingliang, Yu, Xiaofang, Tian, Ruijun
- Journal of chromatography 2019 v.1600 pp. 46-54
- concanavalin A, glycopeptides, glycoproteomics, glycosidases, glycosylation, liquid chromatography, liver, mass spectrometry, mice, packaging materials, peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase, polysaccharides, processing time
- Glycosylation, as a biologically important protein post-translational modification, often alters on both glycosites and glycans, simultaneously. However, most of current approaches focused on biased profiling of either glycosites or glycans, and limited by time-consuming process and milligrams of starting protein material. We describe here a simple and integrated spintip-based glycoproteomics technology (termed Glyco-SISPROT) for achieving a comprehensive view of glycoproteome with shorter sample processing time and low microgram starting material. By carefully integrating and optimizing SCX, C18 and Concanavalin A (Con 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 μg of predigested peptides or directly using 100 μg of protein lysates, in which about 30% 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 is that whole processing time is dramatically reduced from a few days to less than 6 h with good reproducibility when protein lysates were directly processed by Glyco-SISPROT. We expect that this method will be suitable for multi-level glycoproteome analysis of rare biological samples with high sensitivity.