O‑Search-Pattern: A Searching Tool Utilizing the Y‑Ion Pattern to Enhance O‑Glycopeptide Identification for the Analysis of O‑GalNAc Glycoproteome

Different from N-linked glycosylation, the core structures
of mucin
type O-glycans are much more diverse, and the sensitive interpretation
of O-glycopeptide spectra remains a challenge. The Y-ion pattern,
a series of Y-ions with known mass gaps derived from the penta-saccharide
core structure of N-linked glycosylation, is exploited to facilitate
N-glycopeptide identification from their spectra. However, the pattern
of Y ions in O-glycopeptides has not been well studied. In this study,
we found that the Y-ion patterns were also frequently observed in
the spectra of O-glycopeptides, and a special search approach is presented
to identify O-glycopeptides by utilizing the Y-ion patterns. In this
strategy, theoretical O-glycan Y-ion patterns are constructed to match
the experimental Y-ions in O-glycopeptide spectra, which enables the
determination of the mass of some glycans and results in the reduction
of searching space. In addition, a Y-ion pattern-based deisotope process
is also developed to correct the precursor m/z. The new search strategy was applied to search a human
serum data set, and 15.4%–199.0% more O-glycopeptide-spectrum
matches (OGPSMs) and 19.6%–107.1% more glycopeptide sequence
identifications than other state-of-the-art software tools were observed.
This search mode, the O-Search-Pattern, has been implemented into
our database search software, MS-Decipher, and is recommended for
searching the O-glycopeptide spectra acquired by sceHCD (stepped collision
energy higher-energy collisional dissociation).