Optimization And Application Of The Techniques For The Enrichment And Identification Of Sialylated Glycoproteins Based On Titanium Dioxide Materials

Glycosylation is an important post-translational modification of proteins andplays a crucial role in both cellular functions and secretory pathways. Sialic acids(SAs), a family of9carbon-containing acidic monosaccharides, often terminate theglycan structures of cell surface molecules and secreted glycoproteins and perform animportant role in many biological processes. Hence, a more profound profiling of thesialylated glycoproteome is expected to improve our knowledge on this modificationand its effects on protein functions. Blood plasma is an attractive source for thediscovery of biomarkers. Most of the known serum biomarkers are glycoproteins, andthe identification of plasma glycoproteins has been attached increasing importance.In recent years, titanium dioxide (TiO2) chromatography has been widely used toenrich phosphorylated peptides. Due the similar binding property, the use of TiO2forthe enrichment of sialylated glycopeptides has been attempted. In this research, weoptimized the TiO2based approaches for the enrichment of SA glycopeptide. Then theoptimized approach was applied to the identification of SA N-glycopeptides in humanplasma, and several enrichment and fractionation strategies were comparied. Theresults showed that using a combination of a filter-aided sample preparation (FASP)method, TiO2chromatography, multiple enzyme digestion and two-dimensionalreversed-phase peptide fractionation led to a more profound profiling of the SAproteome. In total,982glycosylation sites in413proteins were identified, amongwhich37.8%were newly identified, which established the largest database of sialicacid-containing proteins from human plasma. Using a combination of TiO2chromatography and iTRAQ method, we established the strategy of relativequantitative analysis of N-sialylated glycoproteins. Then this strategy was used for theanalysis of serum of HCC and healthy people and get a result of the differentialproteins between samples.