Status Report on the High-Throughput Characterization of Complex Intact O-Glycopeptide Mixtures

Adam Pap, E. Klement, E. Hunyadi-Gulyás, Z. Darula, Katalin F. Medzihradszky

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

A very complex mixture of intact, human N- and O-glycopeptides, enriched from the tryptic digest of urinary proteins of three healthy donors using a two-step lectin affinity enrichment, was analyzed by LC-MS/MS, leading to approximately 45,000 glycopeptide EThcD spectra. Two search engines, Byonic and Protein Prospector, were used for the interpretation of the data, and N- and O-linked glycopeptides were assigned from separate searches. The identification rate was very low in all searches, even when results were combined. Thus, we investigated the reasons why was it so, to help to improve the identification success rate. Focusing on O-linked glycopeptides, we noticed that in EThcD, larger glycan oxonium ions better survive the activation than those in HCD. These fragments, combined with reducing terminal Y ions, provide important information about the glycan(s) present, so we investigated whether filtering the peaklists for glycan oxonium ions indicating the presence of a tetra- or hexasaccharide structure would help to reveal all molecules containing such glycans. Our study showed that intact glycans frequently do not survive even mild supplemental activation, meaning one cannot rely on these oxonium ions exclusively. We found that ETD efficiency is still a limiting factor, and for highly glycosylated peptides, the only information revealed in EThcD was related to the glycan structures. The limited overlap of results delivered by the two search engines draws attention to the fact that automated data interpretation of O-linked glycopeptides is not even close to being solved. [Figure not available: see fulltext.].

Original languageEnglish
Pages (from-to)1210-1220
Number of pages11
JournalJournal of the American Society for Mass Spectrometry
Volume29
Issue number6
DOIs
Publication statusPublished - Jun 1 2018

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Glycopeptides
Polysaccharides
Throughput
Ions
Search Engine
Search engines
Chemical activation
Complex Mixtures
Lectins
Proteins
Peptides
Molecules
hydronium ion

Keywords

  • EThcD
  • Fragmentation
  • Glycan
  • MS/MS
  • O-Glycopeptide

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

Cite this

Status Report on the High-Throughput Characterization of Complex Intact O-Glycopeptide Mixtures. / Pap, Adam; Klement, E.; Hunyadi-Gulyás, E.; Darula, Z.; Medzihradszky, Katalin F.

In: Journal of the American Society for Mass Spectrometry, Vol. 29, No. 6, 01.06.2018, p. 1210-1220.

Research output: Contribution to journalArticle

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