It is all about the solvent: on the importance of the mobile phase for ZIC-HILIC glycopeptide enrichment

Anal Bioanal Chem. 2017 Jan;409(2):529-538. doi: 10.1007/s00216-016-0051-6. Epub 2016 Dec 1.


Glycopeptide enrichment is a crucial step in glycoproteomics for which hydrophilic interaction chromatography (HILIC) has extensively been applied due to its low bias towards different glycan types. A systematic evaluation of applicable HILIC mobile phases on glycopeptide enrichment efficiency and selectivity is, to date, however, still lacking. Here, we present a novel, simplified technique for HILIC enrichment termed "Drop-HILIC", which was applied to systematically evaluate the mobile phase effect on ZIC-HILIC (zwitterionic type of hydrophilic interaction chromatography) glycopeptide enrichment. The four most commonly used MS compatible organic solvents were investigated: (i) acetonitrile, (ii) methanol, (iii) ethanol and (iv) isopropanol. Glycopeptide enrichment efficiencies were evaluated for each solvent system using samples of increasing complexity ranging from well-defined synthetic glycopeptides spiked into different concentrations of tryptic BSA peptides, followed by standard glycoproteins, and a complex sample derived from human (depleted and non-depleted) serum. ZIC-HILIC glycopeptide efficiency largely relied upon the used solvent. Different organic mobile phases enriched distinct glycopeptide subsets in a peptide backbone hydrophilicity-dependant manner. Acetonitrile provided the best compromise for the retention of both hydrophilic and hydrophobic glycopeptides, whereas methanol was confirmed to be unsuitable for this purpose. The enrichment efficiency of ethanol and isopropanol towards highly hydrophobic glycopeptides was compromised as considerable co-enrichment of unmodified peptides occurred, though for some hydrophobic glycopeptides isopropanol showed the best enrichment properties. This study shows that even minor differences in the peptide backbone and solvent do significantly influence HILIC glycopeptide enrichment and need to be carefully considered when employed for glycopeptide enrichment. Graphical Abstract The organic solvent plays a crucial role in ZIC-HILIC glycopeptide enrichment.

Keywords: Glycopeptide enrichment; Glycopeptide synthesis; Glycoproteomics; HILIC; Hydrophilic interaction chromatography.

MeSH terms

  • Acetonitriles / chemistry
  • Chromatography, Liquid
  • Ethanol / chemistry
  • Glycopeptides / analysis
  • Glycopeptides / chemical synthesis
  • Glycopeptides / isolation & purification*
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Methanol / chemistry
  • Molecular Structure
  • Solvents / chemistry*


  • Acetonitriles
  • Glycopeptides
  • Solvents
  • Ethanol
  • Methanol
  • acetonitrile