An Integrated Strategy For Glycoproteome Analysis

Protein glycosylation is an important post-translational modification,and the site of attachment of a glycan to a protein amino acid is called glycosite.Analysis of glycoproteomics typically involves detection of glycosylation sites,glycan structures,and intact glycopeptides.The processing steps of glycoprotein generally include digestion of proteins,enrichment of glycoproteins or glycopeptides,deglycosylation and so on.However,most of these current approaches focused on biased profiling of either glycosites or glycans,and suffered from time-consuming process and limited by milligrams of starting protein material.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(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.This technology enables largescale proteomic analysis of intact glycopeptides,glycans and glycosites.In total,above 1850 glycosites in ~1770 unique deglycopeptides were characterized from mouse liver by using either 100 ?g of predigested peptides or directly using 100 ?g of protein lysates,in which about 30% of glycosites were released by both PNGase F and Endos.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(single-shot analysis Pearson correlation coefficient > 0.92).To the best of our knowledge,this approach should be one of the most comprehensive glycoproteomic approaches by using limited protein starting material.The experimental results show that Glyco-SISPROT technology is suitable multi-level glycoproteome analysis of rare biological samples with high sensitivity.