Sequest through proteome discoverer

Peptides were first trapped on a C18 column (75 μm inner diameter × 2 cm; nanoViper Acclaim PepMapTM 100, Thermo Scientific) with buffer A (2/98 MeCN/H₂O in 0.1% formic acid) at a flow rate of 4 μL/min over 4 min. Separation was then performed on a 50 cm x 75 μm C18 column (nanoViper Acclaim PepMapTM RSLC, 2 μm, 100 Å, Thermo Scientific) regulated to a temperature of 55 °C with a linear gradient of 5% to 30% of buffer B (100% MeCN in 0.1% formic acid) at a flow rate of 300 nL/min over 100 min. Full-scan MS was acquired in the Orbitrap analyzer with a resolution set to 120,000. Ions from each full scan were HCD fragmented, and analyzed in the linear ion trap. The resulting spectra were then analyzed with Sequest™ through Proteome Discoverer (versions 2.1 or 2.2, Thermo Scientific) by using the SwissProt Homo sapiens Protein Database (022017 and 012018). Carbamidomethyle cysteine (for in-gel digestion only), oxidation of methionine, and N-terminal acetylation were set as variable modifications. Specificity of trypsin digestion was set, 2 missed cleavage sites was allowed for, and mass tolerances in MS and MS/MS was set to 10 ppm and 0.6 Da, respectively. The resulting files were further processed by using myProMS (v 3.5, Ref.^{53 (link)}). The Sequest target and decoy search result were validated with Percolator at 1% false discovery rate (FDR).