Quantitative Proteomics by LC-MS/MS

Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) analysis was performed using quadrupole Orbitrap mass spectrometers, Q-exactive plus (Thermo Fisher Scientific, Waltham, MA, USA), coupled to an Ultimate 3000 RSLC system (Dionex, Sunnyvale, CA, USA) with a nano-electrospray source as previously described, with some modifications [11 (link)]. Peptide samples were separated on a two-column setup with a trap column (300 μm I.D. × 5 mm, C18 3 μm, 100 Å) and an analytical column (75 μm I.D. × 50 cm, C18 1.9 μm, 100 Å). Prior to sample injection, the dried peptide samples were re-dissolved in solvent A (2% acetonitrile and 0.1% formic acid). After the samples were loaded onto the nano LC, a 90-min gradient from 8 to 30% solvent B (100% acetonitrile and 0.1% formic acid) was applied to all samples. The spray voltage was 2.0 kV in positive ion mode, and the temperature of the heated capillary was set to 320 °C. The hyper reaction monitoring (HRM) data-independent acquisition (DIA) method consisted of a survey scan at 35,000 resolution from 400 to 1,220 m/z (AGC target of 3 × 10⁶ or 60-ms injection time). Further, 19 DIA windows were acquired at a resolution of 35,000 with an automatic gain control target of 3e6 and auto injection time [11 (link)]. The stepped collision energy was 10% at 27%.