Utilizing Ion-Pairing Hydrophilic Interaction Chromatography Solid Phase Extraction for Efficient Glycopeptide Enrichment in Glycoproteomics Simon Mysling, † Giuseppe Palmisano, † Peter Højrup,* ,† and Morten Thaysen-Andersen †,‡ Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M, DK-5230, Denmark, and Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales, 2109, Australia Glycopeptide enrichment is a prerequisite to enable structural characterization of protein glycosylation in glycoproteomics. Here we present an improved method for glycopeptide enrichment based on zwitter-ionic hy- drophilic interaction chromatography solid phase extrac- tion (ZIC-HILIC SPE) in a microcolumn format. The method involves TFA ion pairing (IP) to increase the hydrophilicity difference between glycopeptides and non- glycosylated peptides. Three mobile phases were inves- tigated, i.e., 2% formic acid (defined as IP 2% FA ZIC-HILIC SPE), 0.1% TFA and 1% TFA (defined as IP 0.1% TFA and IP 1% TFA ZIC-HILIC SPE) all containing 80% acetoni- trile. Samples of increasing complexities, i.e., digests of single glycoproteins, a five-glycoprotein mixture, and depleted plasma, were used in the study. The presence of TFA in the mobile phase significantly improved the glycopeptide enrichment for all complexities, as evalu- ated by enhanced glycopeptide detection using MALDI- TOF MS and RP-LC-ESI-MS/MS, e.g., the glycopep- tide ion signals were increased by up to 3.7-fold compared to IP 2% FA conditions. The enhanced glyco- peptide detection was promoted by a substantial deple- tion of nonglycosylated peptides, offering an almost complete isolation of IgG glycopeptides using a single SPE enrichment step and a reduction from 711 non- glycosylated peptides observed in the IP 2% FA ZIC- HILIC SPE retained plasma fraction, to only 157 and 97 when 0.1% and 1% TFA was used in the mobile phase. In conclusion, this systematic study has shown that TFA-containing mobile phases increase glycopep- tide enrichment efficiency considerably for a broad range of sample complexities when using ZIC-HILIC SPE. Protein glycosylation is a common post-translational modifica- tion, and the attachment of glycans is known to affect the function of proteins. 1-5 Complete structural characterization of glycopro- teins involves (i) identification of the glycoprotein, (ii) determi- nation of the glycosylation site and the glycan occupancy, and (iii) characterization of the complete set of glycan structures including their monosaccharide composition, linkages, and branch points as well as their relative abundances. Because of the substantial heterogeneity of protein glycosylations, the full char- acterization of even purified glycoproteins can be challenging. However, the large-scale investigation of the glycoproteome, known as glycoproteomics, represents a much greater challenge. 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