Magnetic bead-based hydrophilic interaction liquid chromatography for glycopeptide enrichments.
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 N513 of uromodulin and N470 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.
Department of Biological Science and Technology, National Pingtung University of Science and Technology, Neipu, Pingtung 91201, Taiwan.
Chia-Hao
CH
Yeh
Shu-Hui
SH
Chen
Ding-Tzai
DT
Li
Hong-Ping
HP
Lin
Hung-Jen
HJ
Huang
Chi-I
CI
Chang
Wen-Ling
WL
Shih
Chi-Liang
CL
Chern
Fong-Ku
FK
Shi
Jue-Liang
JL
Hsu
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D060748:Fetuins / Q000378:metabolism; D006020:Glycopeptides / Q000302:isolation & purification* / Q000652:urine; D006031:Glycosylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007553:Isotope Labeling; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D012995:Solubility
D060748:Fetuins; D006020:Glycopeptides; D058185:Magnetite Nanoparticles