Peptide Analysis by Nano-flow LC-MS/MS

Gel tranches were de-stained, with protein reduced, alkylated, digested with trypsin, and peptides extracted and dried^{89 (link)}. Peptides were resuspended in 0.2% formic acid, 0.1% trifluoroacetic acid, and 0.002% zwittergent 3–16 (Calbiochem, San Diego, CA), and analyzed by nano-flow LC–MS/MS using a Q-Exactive Hybrid Quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to a Thermo UltiMate 3000 RSLCnano HPLC system. Peptides were loaded onto a 250 nL OPTI-PAK trap (Optimize Technologies, Oregon City, OR) packed with Michrom Magic C8, 5 µm solid phase (Michrom Bioresources, Auburn, CA). Chromatography was performed using 0.2% formic acid in solvents A (98% water, 2% acetonitrile) and B (80% acetonitrile, 10% isopropanol, 10% water), over a 2–45% B gradient for 60 min at 400 nL/min through a 100 µm × 35 cm PicoFrit column (New Objective, Woburn, MA) packed with Agilent Poroshell 120 EC-C18 (Agilent Scientific Instruments, Santa Clara, CA). MS1 survey scans 350–2000 m/z were acquired at 70,000 resolution targeting 3 × 10⁶ ions and 60 ms maximum inject time, followed by data dependent high energy collisional dissociation MS2 on the top 15 ions at 17,500 resolution targeting 2 × 10⁵ ions with 60 ms maximum inject time, using dynamic exclusion of measured ions for 60 s.