A Label-Free Method Combining Chromatography Enrichment with N-Terminal Specific Proteolysis Enables Sensitive Identification of Tn/sTn Glycosites

Tn (GalNAcα1-O-S/T) and sTn (Neu5Acα2-6GalNAcα1-O-S/T)
antigens, the immature O-glycans, are strongly correlated
with cancer aggressiveness. Tn and sTn antigens exhibit significant
differences between patients and healthy people, and they have emerged
as potential biomarkers for cancer diagnosis and prognosis. Even though
several chemical or chemoenzymatic labeling methods have been developed,
the simultaneous analysis of Tn and sTn glycosites at the proteome
level still faces great challenges. Here, we propose a label-free
method for the sensitive identification of Tn/sTn glycosites from
complex proteome samples. Because Tn/sTn antigens often densely occur
on protein regions rich in Ser/Thr residues but lacking Lys/Arg residues,
the trypsin digestion of these O-glycoproteins will
generate relatively large peptides carrying multiple O-glycans. In this study, large tryptic O-glycopeptides
were specifically enriched using hydrophilic interaction liquid chromatography.
Subsequently, glycopeptides containing a single Tn/sTn antigen were
generated by N-terminal-specific protease digestion, enabling the
identification of Tn/sTn glycosites at the peptide N-termini. We demonstrated
the general applicability of this method to analyze challenging samples,
including HeLa cells and pancreatic ductal adenocarcinoma serum, where
Tn/sTn are low-abundance O-glycans. Furthermore,
a total of 191 Tn/sTn glycosites were identified on 200 glycopeptides
from 136 proteins across two pancreatic cancer cell lines and serum.
In summary, the method is broadly applicable, sensitive and well fitted
to analyze a large cohort of clinical samples.