N-glycan Characterization by Liquid Chromatography Coupled with Fluorimetry and Mass Spectrometry

Human blood plasma and serum have been a source of biomarkers for the indication and progression of many diseases for a few decades now. Human blood plasma is also an excellent source material to enable patients to monitor their health, with a multitude of biomarkers detectable for the assessment of health status. Blood sampling kits are increasingly available for use in the home with no specialist clinical skills required to obtain good quality samples for pathology lab analysis. Many of the proteins that constitute plasma are glycosylated with both N- and O-type glycans. There is increasing interest in the scientific community to identify potential glycan biomarkers or glycan features that are indicative of disease, and in particular disease at an early stage. The quality and reproducibility of glycan analysis data is key in order to identify and utilise glycan-based blood biomarkers with sufficient specificity and sensitivity; hence, the required analytical tools need to be robust. In this chapter, we describe an analytical method for the UHPLC separation of plasma N-glycans which utilizes both glycan reducing terminus fluorophore labeling, to ensure stoichiometric analysis of relative glycan abundance, and online mass spectrometry for glycan identification. Exoglycosidase digestions were employed as example technique to aid and enable structure identification.