EV glycan sample preparation for capillary electrophoresis-laser-induced fluorescence (CE-LIF) analysis

The EV samples used for the capillary electrophoresis-laser-induced fluorescence (CE-LIF) analysis were the same as the ones prepared for the NTA analysis described in the “Nanoparticle tracking analysis (NTA) of EVs using the NanoSight NS300” section. From the 1-mL EV sample, a 20-μL aliquot was used for the EV glycan sample preparation. This 20-μL EV aliquot was dried down in a vacuum concentrator and resuspended in 10 μL of double deionized water (ddiH₂O). The EV samples were processed using the Sciex Fast Glycan Labeling and Analysis Kit (Sciex, cat.#{"type":"entrez-nucleotide","attrs":{"text":"B94499","term_id":"2976836","term_text":"B94499"}}B94499) for denaturation, enzymatic glycan release (PNGase F, 500 NEB units) (New England Biolabs, cat.#P0704), fluorophore labeling (8-aminopyrene-1,3,6-trisulfonate; APTS) (Sciex, cat.#{"type":"entrez-nucleotide","attrs":{"text":"B94507","term_id":"2976844","term_text":"B94507"}}B94507), and excess dye removal by magnetic beads. The CE-LIF analysis was performed using a Beckman Coulter (Sciex) PA800 Plus Pharmaceutical Analysis System equipped with a solid state laser-induced fluorescence detector (λ_ex = 488 nm/λ_em = 520 nm). CE consumables were purchased from Sciex. Analytical reagent grade acetonitrile (ACN) and 2-propanol (IPA) were purchased from Merck KGaA. Ammonium acetate and acetic acid were purchased from Sigma-Aldrich. All separations were carried out using a background electrolyte (BGE) consisting of 7.5 mM ammonium acetate pH 4.5, 10% isopropanol in a 50-cm effective length (60 cm total length), 50 μm ID polyvinyl alcohol (PVA)-coated capillary. The separation voltage was set to 30 kV in reversed polarity mode (cathode at injection side) and the separation temperature was set to 20 °C. Sample injection was performed hydrodynamically by applying 0.5pSi forward pressure for 10 s (~ 9 nL). 32Karat (Sciex, version 10.1) was used for data acquisition and processing. The traces for comparison of the glycan profiles were normalized to the internal standard (maltotriose) and y-axes were scaled to account for the concentration differences obtained by NTA.