Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1224, Issue -, Pages 70-78Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2011.12.057
Keywords
HILIC; Magnetic beads; Glycopeptides enrichment; Glycosylation site; Stable isotope labeling; Glycoproteomics
Funding
- Taiwan NSC [98-2113-M-020-002-MY2]
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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 O-18 enzymatic labeling. (C) 2011 Elsevier B.V. All rights reserved.
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