High-throughput Workflow for Glycan Profiling and Characterisation

Over the last 40 years, the understanding of glycosylation changes in health and disease has evolved significantly and glycans are now regarded as excellent biomarker candidates because of their high sensitivity to pathological changes. However, the discovery of clinical glycobiomarkers has been slow, mainly as a consequence of the lack of high-throughput glycoanalytical workflows that allow rapid glycoprofiling of large clinical sample sets. To generate high-quality quantitative glycomics data in a high-throughput fashion, we have developed a robotised platform for rapid N-glycan sample preparation and glycan characterisation. The sample preparation workflow features a fully automated, rapid glycoprotein affinity purification followed by sequential protein denaturation and enzymatic glycan release on a multiwell ultrafiltration device, thus greatly streamlining all required biochemical manipulations. After glycan purification on solidsupported hydrazide, glycans are fluorescently labelled to allow accurate quantification by ultra-high pressure liquid chromatography (ultra HPLC or UPLC). Subsequent peak assignment can be carried out utilising GlycoBase, a bespoke chromatographic data system developed to aid the analysis of glycans performed using different chromatographic techniques (UPLC, HPLC, Reverse Phase-UPLC, Capillary Electrophoresis).