Abstract
The Tn antigen, an immature truncated O-glycosylation, is a promising biomarker for cancer detection and diagnosis. However, reliable methods for analyzing O-GalNAcylation and complex O-glycosylation are lacking. Here, we develop a novel method, MOTAI, for the sequential analysis of O-glycosylation using different O-glycoproteases. MOTAI conjugates glycopeptides on a solid support and releases different types of O-glycosylation through sequential enzymatic digestion by O-glycoproteases, including OpeRATOR and IMPa. Because OpeRATOR has less activity on O-GalNAcylation, MOTAI enriches O-GalNAcylation for subsequent analysis. We demonstrate the effectiveness of MOTAI by analyzing fetuin O-glycosylation and Jurkat cell lines. We then apply MOTAI to analyze colorectal cancer and benign colorectal polyps. We identify 32 Tn/sTn-glycoproteins and 43 T/sT-glycoproteins that are significantly increased in tumor tissues. Gene Ontology analysis reveals that most of these proteins are ECM proteins involved in the adhesion process of the intercellular matrix. Additionally, the protein disulfide isomerase CRELD2 has a significant difference in Tn expression, and the abnormally glycosylated T345 and S349 O-glycosylation sites in cancer group samples may promote the secretion of CRELD2 and ultimately tumorigenesis through ECM reshaping. In summary, MOTAI provides a powerful new tool for the in-depth analysis of O-GalNAcylation and complex O-glycosylation. It also reveals the upregulation of Tn/sTn-glycoproteins in colorectal cancer, which may provide new insights into cancer biology and biomarker discovery.