C18 analytical column

Mass spectrometry analysis was performed by Carina Sihlbom at the Proteomics Core Facility, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. Samples were analyzed on an Elite mass spectrometer (Thermo Fisher Scientific) interfaced with Easy nLC 1000 liquid chromatography system (Thermo Fisher Scientific). Peptides were separated using an in-house constructed C18 analytical column (300 × 0.075 mm I.D., 3 μm, Dr. Maisch, Germany) using a gradient from 4% to 28% acetonitrile in 0.2% formic acid over 45 min followed by an increase to 80% acetonitrile in 0.2% formic acid for 5 min at a flow of 300 nL/min. Precursor ion mass spectra were acquired at 120K resolution and MS/MS analysis was performed in a data-dependent mode where the 10 most intense precursor ions were selected for fragmentation using CID at a collision energy of 35. Charge states 2 to 4 were selected for fragmentation, and dynamic exclusion was set to 15 s.

LC-MS/MS data acquisition was carried out on a Q Exactive HF mass spectrometer coupled with UltiMate 3000 RSLCnano system (both Thermo Scientific, Carlsbad, CA, USA) [26 (link)]. Peptides were first loaded onto a C18 trap column and then eluted into a self-made C18 analytical column (75 μm × 250 mm, 1.9 μm particle size, 100 Å pore size, Dr. Maisch). Mobile phase A (3% DMSO, 97% H₂O, 0.1% formic acid) and mobile phase B (3% DMSO, 97% ACN, 0.1% formic acid) were used to establish a 60 min gradient. A constant flow rate was set at 300 nL/min. For data-independent acquisition (DIA) analysis, each scan cycle consisted of one full MS scan (R = 60 K, AGC = 3 × 10⁶, max IT = 30 ms, scan range = 350–1250 m/z) followed by 32 variable window DIA MS/MS scans (R = 15 K, AGC = 1 × 10⁶, max IT = 50 ms). HCD collision energy was set to 28.