Glycoproteomics of IgA1: Uncovering Key N-Glycan Composition in Ankylosing Spondylitis: supplementary data (mass spectrometry)

Mass spectrometry (MS) for glycosylation profiling of Fc-derived glycopeptides was conducted in Academia Sinica Glycoscience Core Facility Laboratory (Nankang, Taipei, Taiwan) under the experimental conditions, following the protocol (Medzihradszky 2005). Briefly, IgA reduction was then conducted by adding 1.5 mg/ml dithiothreitol and incubated for 5 min at 95 °C. Reduced samples were then alkylated with 1 mg/ml iodoacetoamide and incubated 20 min at room temperature in the dark then digested by 40 μl of ABC(50 mM ammonium bicarbonate in water) with 0.1 μg trypsin (Promega, sequencing grade) and digested over-night. Samples were acidified with Trifluoroacetic acid to a final concentration of 0.1%. The peptide samples were dried in a SpeedVac evaporator, and stored at -20°C until analysis with LC-MS/MS. IgA1 glycopeptide analysis followed the method described by Yu (Yu et al. 2016). High resolution and high mass accuracy nanoflow LC-MS/MS experiments were performed on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with EASY-nLC 1200 ultra-high pressure system (Thermo Fisher Scientific) and nanoelectrospray ion source (New Objective, Inc.). The digestion solution was injected (3μL) on the self-packed column (C18, 75 μm I.D. × 30 mm, 2.7 μm, 200 Å), and the flow rate was 500 nl/min. The mobile phases consisted of 0.1% formic acid in purified water (mobile phase A) and 0.5% acetic acid and 0.1% formic acid in 80% acetonitrile (mobile phase B). A three-step linear gradient of 5–10% B in 5 min, 10–40% B in 60 min, 40–100% B in 5 min, and 100% B for 10 min was used throughout this study. Survey full-scan MS condition: mass scan range was m/z 375–1500, (AGC target 4E5) with lock mass m/z 445.12003, resolution 100,000 at m/z 400, and maximum injection time of 50 ms. The cycle time 3s were sequentially isolated for CID and detect by trap (AGC target 4E3) and maximum injection time of 300 ms. Electrospray voltage was maintained at 2.2 kV and the capillary temperature was set at 275°C. Fragmentation spectra were recorded with a detection window of m/z 50–2800.