Figure 2

Workflow of N-glycan analysis with UPLC-FLR. Plasma samples were aliquoted into 96 well plates and denatured with sodium dodecyl sulphate (SDS). The plate was sealed and incubated at 65 °C for 10 min. IGEPAL CA-630 was added and sample mixed by pipetting up and down. This was then followed by incubation at room temperature. Glycans were freed from their bound glycoproteins by adding peptide N-glycosidase F (PN-Gase F) and incubation at 37 °C for 18 h. glycans were then fluorescently labelled with 2-aminobenzamide and incubated for 2 h at 65 °C. This was followed by four-step washing procedure with acetonitrile and 2AB glycans were eluted using ultra-pure water. Samples were injected into the UPLC and analysed under the following conditions: solvent A = 100 Mm ammonium formate, solvent B = acetonitrile, flow rate 0.1 ml/min, pH = 4.4. Structural assignments and normalisation of glycan peaks were then performed.