Magnetic bead-based hydrophilic interaction liquid chromatography for glycopeptide enrichments

Chia Hao Yeh, Shu-Hui Chen, Ding Tzai Li, Hong-Ping Lin, Hung Jen Huang, Chi I. Chang, Wen Ling Shih, Chi Liang Chern, Fong Ku Shi, Jue Liang Hsu

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47 Citations (Scopus)


Purification of glycopeptides prior to the analysis by mass spectrometry (MS) is demanded due to ion suppression effect during ionization caused by the co-presence of non-glycosylated peptides. Among various purification methods, hydrophilic interaction liquid chromatography (HILIC) has become a popular method in recent years. In this work, we reported a novel magnetic bead-based zwitterionic HILIC (ZIC-HILIC) material which was fabricated by coating a zwitterionic polymer synthesized by spontaneous acid-catalyzed polymerization of 4-vinyl-pyridinium ethanesulfonate monomer on iron oxide magnetic nanoparticles. The resulting magnetic ZIC-HILIC nanoparticles were shown to provide high specificity and high recovery yield (95-100%) for the enrichment of glycopeptides from a standard glycoprotein, fetuin, using a simple magnetic bar. In addition, we proposed a two-step HILIC enrichment strategy using magnetic ZIC-HILIC nanoparticles for a large scale analysis of glycoproteins in complex biological samples. Using this approach, we identified 85 N-glycosylation sites in 53 glycoproteins from urine samples. Two novel glycosylation sites on N 513 of uromodulin and N 470 of lysosomal alpha-glucosidase which have not yet been reported were identified by two-step HILIC approach. Furthermore, all these identified sites were confirmed by studies conducted using PNGase F deglycosylation and 18O enzymatic labeling.

Original languageEnglish
Pages (from-to)70-78
Number of pages9
JournalJournal of Chromatography A
Publication statusPublished - 2012 Feb 10

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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    Yeh, C. H., Chen, S-H., Li, D. T., Lin, H-P., Huang, H. J., Chang, C. I., Shih, W. L., Chern, C. L., Shi, F. K., & Hsu, J. L. (2012). Magnetic bead-based hydrophilic interaction liquid chromatography for glycopeptide enrichments. Journal of Chromatography A, 1224, 70-78.