Abstract
Sialyl Thomsen-Friedenreich (ST) antigen, the attachment of trisaccharide Neu5Acα2-3Galβ1-3GalNAc on a serine or threonine residue, is a well-known tumor-associated carbohydrate antigen. There is a growing body of studies indicating that aberrant expression of ST antigen has a strong relationship with tumor invasion and metastasis. However, details about the biological functions of ST antigen are still poorly understood, as there is a lack of an effective method for glycosylation site analysis of the ST antigen. In this study, we developed a chemoenzymatic labeling strategy for the efficient site-specific analysis of ST antigen. A temperature-sensitive probe carried by the CMP-Neu5Ac derivative was designed and synthesized. The probe could be specifically introduced to ST antigen through a one-pot reaction mediated by sialyltransferase ST6GalNAc4. Controlled increase-reduction temperature cycling enabled the effective enrichment of the labeled glycopeptides from complex biological samples. Subsequently, the enriched glycopeptides were released via UV356 irradiation for mass spectrometry analysis, providing the details of where ST antigen attaches to proteins. Finally, this approach was successfully applied to globally profile ST antigen-modified proteins in three breast cancer cell lines: MCF7, MDA-MB-231, and MDA-MB-468.