Higher-Energy Collisional Dissociation Mass Spectrometry Fragmentation Enables Distinguishing O‑GlcNAc from Tn Antigen in Cancer Cells

Protein glycosylation plays essential roles in various
biological
processes, and thus determining the glycan structure present on the
protein is essential to comprehensively understand these events. However,
distinguishing saccharide stereoisomers is challenging, especially
when their structures are very similar and their molecular weight
and potential glycosylation sites are identical. One representative
example is O-linked β-N-acetylglucosamine (O-GlcNAc)
and O-linked α-N-acetylgalactosamine (Tn antigen).
Traditional biochemistry approaches used in separating O-GlcNAc- and
Tn antigen-modified peptides mainly include chemical derivatizations,
lectins, and antibodies. However, subsequent mass spectrometry (MS)
analysis is still required if one aims to determine the exact glycosylation
site. Herein, a straightforward approach using the ratio of relative
abundance (RA) of two fragment ions (RA126.055/RA138.055) in higher-energy collisional dissociation (HCD) MS without relying
on the traditional biochemistry technique is reported to discriminate
between O-GlcNAc and Tn antigen. This ratio was verified by synthetic
glycopeptides and proteomic analysis in HeLa cells, where 10 proteins
were found to be O-GlcNAcylation and 4 proteins were found to be Tn
antigen-modified. Overall, this method can be extensively employed
in liquid chromatography–mass spectrometry (LC-MS)-based proteomic
studies and thus is of importance in biological and biomedical research.