Oral Presentation 23rd Annual Lorne Proteomics Symposium 2018

SWATH analysis of human plasma glycopeptides without predefined glycan compositional knowledge (#54)

Chi-Hung Lin 1 2 3 , Christoph Krisp 1 2 , Nicolle H Packer 1 3 , Mark P Molloy 1 2
  1. Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia
  2. Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW, Australia
  3. Institute for Glycomics, Griffith University, Southport, QLD, Australia

Glycoproteomics investigates glycan moieties in a site specific manner to reveal the functional roles of protein glycosylation. Identification of glycopeptides from data-dependent acquisition (DDA) relies on high quality MS/MS spectra of glycopeptide precursors and often requires manual validation to ensure confident assignments.To explore alternative acquisition strategies, we investigated the utilities of pseudo-MRM using MRM_HR and data independent acquisitions using SWATH for glycopeptide analysis. These approaches allow data acquisition over the full MS/MS scan range allowing data re-analysis post-acquisition, without data re-acquisition. The advantage of MRM-HR over DDA for N-glycopeptide detection was demonstrated from targeted analysis of bovine fetuin where all three N-glycosylation sites were detected, which was not the case with DDA. To overcome the duty cycle limitation of MRM-HR acquisition needed for analysis of complex samples such as plasma we trialed DIA. This allowed development of a targeted DIA method to identify N-glycopeptides without pre-defined knowledge of the glycan composition, thus providing the potential to identify N-glycopeptides with unexpected structures. This workflow was demonstrated by detection of 59 N-glycosylation sites from 41 glycoproteins from a HILIC enriched human plasma tryptic digest. 21 glycoforms of IgG1 glycopeptides were identified including two truncated structures that are rarely reported.