Tryptic peptides were desalted (Rappsilber et al., 2007 (link)) and separated by reverse phase nano-LC-MS/MS (column: 12 cm/75um C18 built-in-emitter column, Nikkyo Technos Co., Ltd. Japan, EasyLC 1200, Thermo Scientific) using a 70-min analytical gradient, increasing from 2% B/98% A to 38%B/62% A (A: 0.1% formic acid, B: 80% Acetonitrile/0.1% formic acid) at 300 nL/min. The mass spectrometer (Fusion Lumos, Thermo Scientific) was operated in high/high mode (120,000 and 30,000 for MS1 and MS2, respectively). Auto Gain Control was set at 50,000 for MS2. MS1 scan range was set to m/z 375–1500 and m/z 110 was set as lowest recorded mass in MS2. One-point lock mass calibration was used. All data were quantified and searched against a Uniprot mouse database using MaxQuant (v. v. 1.6.0.13) (Cox et al., 2014 (link)). Oxidation of methionine and protein N-terminal acetylation were allowed as variable modifications, cysteine carbamidomethyl was set as a fixed modification, and two missed cleavages were allowed. The ‘match between runs’ option was enabled, and false discovery rates for proteins and peptides were set to 1%. Protein abundances measured using label free quantitation (Tyanova et al., 2016 (link)).
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