Capture and Analysis of Cell Surface N-Glycans by Hydrazide-Modified Magnetic Beads and CE-LIF

Abstract

Cell surface proteins are important target molecules for biomarkers discovery. Diseases are often reflected within the glycan profiles of proteins. As such, studying the glycan profiles of cell surface proteins may give rise to new biomarker or drug target discoveries. Focused on cell surface glycoproteins, hydrazine technology had been applied to cell surface protein capturing (CSC) technology. In this study, hydrazide-functionalized magnetic beads were used to capture surface glycoproteins on intact cells and the released N-glycan profiling, except protein profiling, was investigated by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. The use of magnetic beads gave rapid separation and enrichment of the captured glycoproteins. The sample processing parameters, including the ratio of cells to hydrazine groups, oxidation time and cell incubation time with magnetic beads, cell lysis and glycopeptide cleavage methods, were evaluated using MALDI-TOF–TOF mass spectrometry analysis. Subsequently, citric acid was applied at optimized temperature for the glycan cleavage from the beads. The technique developed has the potential in sensing, diagnostic and therapeutic applications by speeding up the method development of glycoprotein identification, N-glycan profiling, drug target screening and biomarker discovery.