LC-MS analysis of procainamide-labeled N-glycans

Samples were analyzed by using HILIC-LC on an Ultimate 3000 UHPLC with a BEH-Glycan 1.7 μm, 2.1 × 150 mm column (Waters, Milford, Mass) at 40°C with a fluorescence detector (λex = 310 nm, λem = 370 nm) controlled by Bruker HyStar 3.2 (Bruker, Billerica, Mass). Buffer A was 50 mmol/L ammonium formate made from LudgerSep N Buffer stock solution, pH 4.4 (LS-N-BUFFX40); buffer B was acetonitrile (acetonitrile 190 far UV/gradient quality; Romil #H049). Samples were injected in 25% aqueous/75% acetonitrile; injection volume was 25 μL. Separation was performed with a linear gradient of 76-53% ACN at 0.4 mL/min in a 71-minute analytic run. PROC-labeled glucose homopolymer was used as a system suitability standard, as well as an external calibration standard, for glucose unit allocation for the system. Chromeleon Data Software (version 7.2; Thermo Fisher Scientific, Waltham, Mass) with a cubic spline fit was used to allocate glucose unit values to peaks. A Bruker amaZon Speed ETD electrospray mass spectrometer (Bruker) was coupled directly after the ultra-HPLC with fluorescence detection without splitting. The instrument scanned samples in maximum resolution mode with a positive ion setting, mass spectrometry (MS) plus 3 MS/MS scans, nebulizer pressure of 14.5 psi, nitrogen flow of 10 L/min, and capillary voltage of 4500 V. MS/MS was performed on 3 ions in each scan sweep with a mixing time of 40 ms. MS data were analyzed by using Bruker Compass DataAnalysis 4.1 software.