Poster Presentation 23rd Annual Lorne Proteomics Symposium 2018

Enrichment of lowly-hydrophilic truncated N-glycopeptides using IP-HILIC-SPE (#116)

Ian Loke 1 , Harry Tjondro 1 , Katalin F. Medzihradszky 2 , Morten Thaysen-Andersen 1
  1. Biomolecular Discovery Research Centre, Macquarie University, Sydney, NSW, Australia
  2. UCSF Mass Spectrometry Facility, University of California, San Francisco, San Francisco, CA

Unbiased enrichment of intact glycopeptides from complex peptide mixtures is important to facilitate deep and quantitative glycoprofiling in bottom-up glycoproteomics. Ion-pairing zwitter-ionic hydrophilic interaction liquid chromatography (IP-ZIC-HILIC) SPE provides efficient bulk enrichment of N-glycopeptides due to their common highly polar conjugated N-glycans1. With the discovery of lowly-hydrophilic N-glycans in the mammalian glycoproteome spanning the highly truncated paucimannosidic (Man1-3GlcNAc2Fuc0-1)2 and chitobiose core (GlcNAc1-2Fuc0-1)3,4 type structures, it becomes important to investigate if the corresponding glycopeptides are discriminated against in IP-ZIC-HILIC SPE. We investigated this aspect by first generating relative simple tryptic and non-tryptic mixtures of human glycopeptides carrying a spectrum of glycoforms including chitobiose, paucimannosidic and complex N-glycans. The glycopeptides were profiled before and after enrichment using various stationary/mobile phase conditions and column capacities. The resulting LC-MS/MS data was investigated for qualitative and quantitative bias. Tryptic and non-tryptic paucimannosidic peptides including the short Man1GlcNAc2-peptides appeared to be quantitatively retained whereas chitobiose core type-peptides, in particular the GlcNAc1-peptides were often under-represented in the retentate. IP-ZIC-HILIC-retained tryptic glycopeptides in highly complex mixtures were then investigated relative to matching non-enriched experiments using Byonic-based identification, which supported that tryptic paucimannosidic peptides are often fully retained regardless of the peptide carrier. Finally, in silico calculations of the relative hydrophilicity (ΔGoctanol:water) of confidently identified chitobiose core and paucimannosidic intact glycopeptides were performed using published glycoproteomics datasets. These multiple approaches provide valuable insight into the retention ability of IP-ZIC-HILIC SPE for truncated lowly-hydrophilic N-glycopeptides. We conclude that tryptic paucimanosidic peptides appear to be well-retained under optimised IP-ZIC-HILIC SPE conditions whereas quantitative chitobiose-peptide retention is peptide carrier dependent and thus risk being under-represented in IP-ZIC-HILIC SPE-based glycoproteomics.

[1] Mysling et al. Anal Chem. 2010. 82(13):5598.

[2] Loke et al. Mol Cell Proteomics. 2017. 16(8):1507.

[3] Trinidad et al. Mol Cell Proteomics. 2013. 12(12):3474.

[4] Loke et al. Biomolecules. 2015. 5(3):1832.