Symmetry c18 180 m 20 mm trapping column

Quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed in singlicate on 0.3 µg of protein digest per sample, and the pool was analyzed four times with 0.3 μg injections spaced evenly across the run queue using the acquisition method immediately described below. The method used liquid chromatography on a nanoACQUITY UPLC system (Waters Corp, Milford, MA) coupled to a Q-Exactive Plus high-resolution accurate mass tandem mass spectrometer (Thermo Fisher Scientific) with nanoelectrospray ionization. Mobile phase A and B consisted of 0.1% formic acid (v/v) in water and acetonitrile, respectively. Peptides were first trapped at 5 µl/min, 99.9%A, on a 5 µm Symmetry C18 180 µm × 20 mm trapping column (Waters Corp). Analytical separations were performed on a 1.8 µm Acquity HSS T3 C18 75 µm × 250 mm column (Waters Corp) using a linear gradient of 5–40% B over 90 min, at a flow rate of 0.4 µl/min and column temperature of 55 °C. Data collection on the Q-Exactive Plus mass spectrometer were performed in data-dependent acquisition mode of acquisition with Rs = 70,000 (@ m/z 200) full MS scan from m/z 375 to 1600 with a target AGC value of 1e6 ions followed by 10 MS/MS scans at Rs = 17,500 (@ m/z 200) at a target AGC value of 5e4 ions. A 20 s dynamic exclusion was employed.