Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Enrichment of Lowly-Hydrophilic N- and O-glycopeptides using Ion-Pairing ZIC-HILIC-SPE (#703)

Ian Loke 1 , Harry Tjondro 2 , Rebeca Sakuma 2 , Katalin Medzihradszky 3 , Morten Thaysen-Andersen 2
  1. Department of Biological Sciences, National University of Singapore, Singapore
  2. Department of Molecular Sciences, Maccquarie University, Sydney, New South Wales, Australia
  3. UCSF Mass Spectrometry Facility, University of California, San Francisco, California, USA

Trifluoroacetic acid-based Ion-pairing zwitterionic hydrophilic interaction liquid chromatography solid phase extraction (TFA-IP-ZIC-HILIC-SPE) is a key sample processing step in quantitative glycoproteomics that provides unbiased enrichment of common N-glycopeptides from complex peptide mixtures1. With the discovery of truncated and lowly-hydrophilic N-glycans in the mammalian glycoproteome spanning the paucimannosidic (Man1-3GlcNAc2Fuc0-1)2 and chitobiose core (GlcNAc1-2Fuc0-1)3,4 type structures, it becomes important to investigate if such N-glycopeptides and O-glycopeptides are efficiently enriched using TFA-IP-ZIC-HILIC-SPE. We here investigate this by performing quantitative LC-MS/MS profiling of various tryptic and non-tryptic peptide mixtures containing human N- and O-glycopeptides carrying paucimannosidic, chitobiose and O-GlcNAc moieties using ZIC-HILIC-SPE enrichment with formic acid (FA) and TFA as mobile phase additives. The paucimannosidic peptides including the lowly hydrophilic Man1GlcNAc2-peptides were quantitatively retained using TFA, but the even less hydrophilic chitobiose core peptides, in particular the GlcNAc1-peptides, were often under-represented. Interestingly, using the less hydrophobic ion-pairing reagent FA, both paucimannosidic and chitobiose core peptides were more often quantitatively retained. The O-GlcNAc peptide profiling recapitulated these trends by demonstrating dramatically better retention using FA-IP-ZIC-HILIC-SPE than under TFA condition. The comparatively better retention of lowly hydrophilic glycopeptides with FA-IP-ZIC-HILIC-SPE was rationalised using in silico calculations of the peptide hydropathy (ΔGoct-water) identified relative to published and putative glycopeptides in the human and mouse glycoproteomes, which suggest a significant peptide carrier influence on their retention propensity. In conclusion, we here show that paucimannosidic peptides, but not necessarily chitobiose core peptides, generally are well-retained when using the conventional TFA-IP-ZIC-HILIC-SPE enrichment whereas the less used FA-IP-ZIC-HILIC-SPE appears to be a universally better strategy for quantitative glycoproteomics of mixtures containing lowly-hydrophilic glycopeptides.

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