ac8b04184_si_007.xlsx (45.7 kB)
A New Searching Strategy for the Identification of O‑Linked Glycopeptides
dataset
posted on 2019-02-25, 00:00 authored by Jiawei Mao, Xin You, Hongqiang Qin, Chengye Wang, Liming Wang, Mingliang YeFor the analysis
of homogeneous post-translational modifications
such as protein phosphorylation and acetylation, setting a variable
modification on the specific residue(s) is applied to identify the
modified peptides for database searching. However, this approach is
often not applicable to identify intact mucin-type O-glycopeptides
due to the high microheterogeneity of the glycosylation. Because there
is virtually no carbohydrate-related tag on the peptide fragments
after the O-glycopeptides are dissociated in HCD, we find it is unnecessary
to set the variable mass tags on the Ser/Thr residues to identify
the peptide sequences. In this study, we present a novel approach,
termed as O-Search, for the interpretation of O-glycopeptide HCD spectra.
Instead of setting the variable mass tags on the Ser/Thr residues,
we set variable mass tags on the peptide level. The precursor mass
of the MS/MS spectrum was deducted by every possible summed mass of
O-glycan combinations on at most three S/T residues. All the spectra
with these new precursor masses were searched against the protein
sequence database without setting variable glycan modifications. It
was found that this method had much decreased search space and had
excellent sensitivity in the identification of O-glycopeptides. Compared
with the conventional searching approach, O-Search yielded 96%, 86%,
and 79% improvement in glycopeptide spectra matching, glycopeptide
identification, and peptide sequence identification, respectively.
It was demonstrated that O-Search enabled the consideration of more
glycan structures and was fitted to analyze microheterogeneity of
O-glycosylation.