Linkage-specific characterization of sialylated n-glycans from human plasma by lc-ms

S3.3 Glycomics
Location (hall): 
Start/end time: 
Monday, July 1, 2019 - 18:00 to 18:15

Alan Moran1,2, Richard Gardner2, Jennifer Hendel2, Daniel I.R. Spencer2

1Leiden University Medical Center, Leiden, The Netherlands, 2Ludger Ltd., Abingdon, United Kingdom

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 [1]. In these cases, aberrant sialylation can be analysed using several methods including liquid chromatography-mass spectrometry (LC-MS) and MALDI-MS [2]. Various sialic acid chemical modification techniques have been applied in MALDI-MS analyses in order to allow linkage-specific characterization [3]. In comparison, sialic acid linkage differentiation using LC is often based on retention times and/or sequential exoglycosidase digestions [4]. However, few LC-MS methods exist that allow linkage-specific characterization of sialic acids [5].

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) [6]. Next, ethyl esterification and amidation of sialic acids was carried out with a subsequent GHP membrane clean-up [3]. 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.

Figure 1: Extracted ion chromatograms of procainamide labelled A2G2S2 N-glycan from human plasma. LC-MS analysis was performed using hydrophilic interaction liquid chromatography mass spectrometry (HILIC-MS) with a (Waters) BEH-glycan column. Top: Underivatized N-glycan is shown. Bottom: Display of derivatized N-glycan(s) for α(2,6) α(2,6) (left), α(2,3)α(2,6) (middle), and α(2,3)α(2,3) (right).

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