A Chemoenzymatic Labeling Strategy for Site-Specific Analysis of Tumor-Associated Sialyl Thomsen–Friedenreich Antigen

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.