posted on 2018-10-16, 00:00authored byXin You, Yating Yao, Jiawei Mao, Hongqiang Qin, Xinmiao Liang, Liming Wang, Mingliang Ye
Human serum is a
complex body fluid that contains various N-linked
and O-linked glycoproteins. Compared with N-linked glycoproteins,
the serum O-linked glycoproteins are not well-studied due to their
high heterogeneity and their low abundance. Herein, we presented a
novel chemoenzymatic method to analyze core-1 type of O-GalNAcylation
in human serum. In this approach, the tryptic digest of serum was
first subjected to PNGase F treatment to release the N-glycan and
was then treated with strong acid to release sialic acid residues
from mucin-type O-glycans. In this way, the internal Gal/GalNAc residues
were exposed and were oxidized by the galactose oxidase to carry the
aldehyde groups. The oxidized O-GalNAcylated peptides were then captured
by hydrazide beads and eluted with methoxylamine for LC–MS/MS
analysis. The de-N-deglycosylation decreased the abundance of N-glycopeptides,
the desialylation simplified the O-glycans and the enzymatic oxidization
conferred the enrichment specificity. We have demonstrated that this
method was fitted to analyze O-GalNAcylated peptides with high confidence.
This method was applied to analyze human serum, which resulted in
the identification of 59 O-GalNAc modified peptide sequences corresponding
to 38 glycoproteins from 50 μL of serum. This method is expected
to have broad applications in the analysis of O-glycoproteome.