A “One-Step” Strategy for the Global Characterization of Core-Fucosylated Glycoproteome

Core fucosylation, a special type of N-linked
glycosylation, is important in tumor proliferation, invasion, metastatic
potential, and therapy resistance. However, the core-fucosylated glycoproteome
has not been extensively profiled due to the low abundance and poor
ionization efficiency of glycosylated peptides. Here, a “one-step”
strategy has been described for protein core-fucosylation characterization
in biological samples. Core-fucosylated peptides can be selectively
labeled with a glycosylated probe, which is linked with a temperature-sensitive
poly(N-isopropylacrylamide) (PNIPAM) polymer, by
mutant endoglycosidase (EndoF3-D165A). The labeled probe can be further
removed by wild-type endoglycosidase (EndoF3) in a traceless manner
for mass spectrometry (MS) analysis. The feasibility and effectiveness
of the “one-step” strategy are evaluated in bovine serum
albumin (BSA) spiked with standard core-fucosylated peptides, H1299,
and Jurkat cell lines. The “one-step” strategy is then
employed to characterize core-fucosylated sites in human lung adenocarcinoma,
resulting in the identification of 2494 core-fucosylated sites distributed
on 1176 glycoproteins. Further data analysis reveals that 196 core-fucosylated
sites are significantly upregulated in tumors, which may serve as
potential drug development targets or diagnostic biomarkers. Together,
this “one-step” strategy has great potential for use
in global and in-depth analysis of the core-fucosylated glycoproteome
to promote its mechanism research.