The developing era of precision medicine requires novel disease biomarkers that have molecular characteristics with predictive and/or prognostic value. For example, molecules such as α(2,3)- and α(2,6)- linked sialic acid on N-glycans have been implicated in studies investigating malignant transformation . In these cases, aberrant sialylation can be analysed using several methods including liquid chromatography-mass spectrometry (LC-MS) and MALDI-MS . Various sialic acid chemical modification techniques have been applied in MALDI-MS analyses in order to allow linkage-specific characterization . In comparison, sialic acid linkage differentiation using LC is often based on retention times and/or sequential exoglycosidase digestions . However, few LC-MS methods exist that allow linkage-specific characterization of sialic acids .
A novel experimental approach for sialic acid linkage differentiation will be presented that involves the selective chemical derivatization of sialylated N-glycans followed by analysis using LC-MS. First, PNGase-F released N-glycans from human plasma were fluorescently labelled using procainamide, followed by removal of excess labelling reagent using a hydrophilic interaction chromatography (HILIC)-based technique (GHP) . Next, ethyl esterification and amidation of sialic acids was carried out with a subsequent GHP membrane clean-up . Finally, modified N-glycans were analysed by HILIC-MS using a (Waters) BEH-glycan column.
There were prominent differences in the retention times associated with derivatized vs. non-derivatized N-glycans (figure 1). In general, derivatization resulted in overall earlier elution of the sialylated glycans whereby glycans with α(2,6)-linkages were most effected. The final assignment of derivatized sialylated structures was carried out based on three main features: retention times specific to sialic acid linkage, parent ion mass and specific molecular ions. As a result, a more in-depth characterization of human plasma N-glycans could be made. Thus, this study supports the use of chemical modification followed by LC-MS analysis as a platform to characterise α2,3- and α2,6-sialylated glycan isomers. Importantly, this may have implications for precision medicine whereby abnormal changes in these monosaccharides may represent a potential new class of disease biomarker.
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