UPLC-MS Proteomics Workflow for Protein Profiling

UPLC-MS was performed as described previously [15 (link)]. Briefly, four μL samples (equivalent to ~1.4 μg total protein) were injected to nano Acquity UPLC (Ultra Performance Liquid Chromatography)–system (Waters Corporation, MA, USA). TRIZAIC nanoTile 85 μm × 100 mm HSS-T3u wTRAP was used as separation device. Samples were loaded, trapped and washed for two minutes with 8.0 μL/min with 1% B. The analytical gradient used is as follows: 0–1 min 1% B, at 2 min 5% B, at 65 min 30% B, at 78 min 50% B, at 80 min 85% B, at 83 min 85% B, at 84 min 1% B and at 90 min 1% B with 450 nL/min. Buffer A was 0.1% formic acid in water and buffer B was 0.1% formic acid in acetonitrile. Data were acquired using HDMSE mode with Synapt G2-S HDMS (Waters Corporation, MA, USA). Data was collected in the range of 100–2000 m/z, scan time one-second, IMS wave velocity 650 m/s. Collision energy was ramped from 20 to 60 V. Calibration was performed with Glu1-Fibrinopeptide B MS2 fragments. Glu1-Fibrinopeptide B precursor ion was used as a lock mass during the runs. The samples were run in triplicates.