Open AccessMass Spectrometric Mapping of Glycoproteins Modified by Tn-Antigen Using Solid-Phase Capture and Enzymatic Release
Author(s) -
Weiming Yang,
Minghui Ao,
Angellina Song,
Yuanwei Xu,
Lori J. Sokoll,
Hui Zhang
Publication year - 2020
Publication title -
analytical chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.117
H-Index - 332
eISSN - 1520-6882
pISSN - 0003-2700
DOI - 10.1021/acs.analchem.0c01564
Subject(s) - glycosylation , glycoprotein , chemistry , glycan , enzyme , biochemistry , glycosyltransferase , antigen , monosaccharide , microbiology and biotechnology , biology , immunology
Tn-antigen (Tn), a single N -acetylgalactosamine (GalNAc) monosaccharide attached to protein Ser/Thr residues, is found on most cancer yet rarely detected in adult normal tissues as reported in previous studies, featuring it as one of the most distinctive signatures of cancer. Although it is important in cancer, Tn modified glycoproteins are not entirely clear owing to the lack of a suitable method. Knowing the Tn-glycosylated proteins and glycosylation sites are essential to the prevention, diagnosis, and therapy of cancer associated with the expression of Tn. Here, we introduce a method named EXoO-Tn for large-scale mapping of Tn-glycosylated proteins and glycosylation sites. EXoO-Tn utilizes solid-phase immobilization of proteolytic peptides of proteins, which modifies Tn by glycosyltransferase C1GalT1 with isotopically labeled UDP-Gal( 13 C 6 ), to tag and convert Tn to Gal( 13 C 6 )-Tn, which gives rise to a unique glycan mass. The exquisite Gal( 13 C 6 ) modified Tn are then recognized by a human-gut-bacterial enzyme, OpeRATOR, and released at the N-termini of the Gal( 13 C 6 )-Tn-occupied Ser/Thr residues from immobilized peptides to yield site-containing glycopeptides. The effectiveness of EXoO-Tn was benchmarked by analyzing Jurkat cells, where 947 Tn-glycosylation sites from 480 glycoproteins were mapped. The EXoO-Tn was further applied to the analysis of pancreatic cancer sera, where Tn-glycoproteins were identified. Given the significance of Tn in cancer, EXoO-Tn is anticipated to have broad translational and clinical utilities.