High-Throughput Analysis of the Plasma N-Glycome by UHPLC
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The understanding of glycosylation alterations in health and disease has evolved significantly and glycans are considered to be relevant biomarker candidates. High-throughput analytical technologies capable of generating high-quality, large-scale glycoprofiling data are in high demand. Here, we describe an automated sample preparation workflow and analysis of N-linked glycans from plasma samples using hydrophilic interaction liquid chromatography with fluorescence detection on an ultrahigh-performance liquid chromatography (UHPLC) instrument. Samples are prepared in 96-well plates and the workflow features rapid glycoprotein denaturation, enzymatic glycan release, glycan purification on solid-supported hydrazide, fluorescent labeling, and post-labeling cleanup with solid-phase extraction. The development of a novel approach for plasma N-glycan analysis and its implementation on a robotic platform significantly reduces the time required for sample preparation and minimizes technical variation. It is anticipated that the developed method will contribute to expanding high-throughput capabilities to analyze protein glycosylation.
Key wordsN-linked glycosylation Plasma Glycan analysis Ultrahigh-performance liquid chromatography Robotics Automation High-throughput
The authors acknowledge support from the European Union FP7 GastricGlycoExplorer ITN under grant agreement no. 316929. and HighGlycan under grant agreement no. 278535. We would like to acknowledge Dr. Karol Polom, Dr. Giovanni Corso, and Dr. Franco Roviello from University Hospital of Siena for providing gastric cancer serum samples. The UHPLC instrument was obtained with a grant from the IngaBritt and Arne Lundberg’s Research Foundation.
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