Mass spectrometry

Excised protein bands were destained by washing with ammonium bicarbonate and then 50% acetonitrile in ammonium bicarbonate. Gel pieces were subjected to reduction and alkylation with dithiothreitol and iodoacetamide, respectively. For digestion, sequencing-grade modified trypsin (Promega) was added at 1:50 w/w (enzyme: protein) and the reaction was incubated at 37 °C overnight. The reaction was stopped by addition of 1/10 volume 10% formic acid. The product was transferred to glass autosampler vials for LC/MS analysis. Peptide separation was performed on a Nanoflow Dionex 3000 RSLC system using C18 EASY-spray™ column (P/N ES803, C18 2 µm, 100 A, 75 µm × 50 cm; Thermo Scientific), maintained at 50 °C. Chromatographic separation was achieved with an acetonitrile gradient in water (2–80 %) over 100 min, using 0.1% w/v formic acid as the ion-pairing agent. The trapping column was a C18 PepMap^TM 100, 5 µm 100 A (Dionex^TM) and was maintained at 45 °C. Data acquisition was performed in data-dependent analysis mode on a Q-exactive plus system (Thermo Scientific). Full scan MS¹ was performed at 70,000 MS resolution with an automatic gain control of 1e⁶ and injection time of 100 ms. The scan mass range was 375 to 1400 m/z. The ten most abundant top ions were selected for MS/MS analysis with a normalized collision energy of 30. MS² data acquisition was performed at 17,500 MS resolution with an automatic gain control of 1e⁵ and a maximum injection time of 100 ms. The isolation window was set to 1.3 m/z with an under fill ratio of 0.4%. Dynamic exclusion was set to 15 s and the full-width at half-maximum (FWHM) was 5 s.