Selecting proper and efficient glycopeptide enrichment approaches are essential for mass spectrometry-based glycoproteomics since glycopeptides are usually with microheterogeneity and low abundance in most biological samples. Herein, we introduced a cotton hydrophilic interaction liquid chromatography (HILIC) approach for large-scale glycopeptide enrichment with 80% acetonitrile/1% trifluoroacetic acid as the optimal sample loading buffer. The comparison of cotton HILIC with Venusil HILIC and mixed anion-exchange (MAX) approaches indicated that cotton HILIC was superior in overall glycopeptide enrichment, whereas Venusil HILIC preferred in complex glycan structures and MAX performed better with high mannose glycans. Exploration of capacity and recovery rate of cotton HILIC illustrated that 5mg cotton packed in a 200μL tip achieved a reasonable glycopeptide enrichment performance (~6% recovery) from ~0.5mg peptides. In conclusion, cotton HILIC can be used as an optional glycopeptide enrichment approach in glycosylation analysis with its specific merit.

College of Life Science Northwest University Xi'an Shaanxi Province 710069 People's Republic of China

Department of Medical Genetics Xi'an 4th Hospital Xi'an Shaanxi Province 710004 People's Republic of China

South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses Research Institute of Fish Culture and Hydrobiology Faculty of Fisheries and Protection of Waters University of South Bohemia in Ceske Budejovice 38925 Vodnany Czech Republic

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