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.
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