Orbitrap q exactive hf x spectrometer

Cells were treated with a combination of SAS and PAG for 72 h. Dry, PBS-washed frozen cell pellets were lysed directly in 8 M urea, 50 mM ammonium bicarbonate supplemented with 1 × Complete protease inhibitor (Roche), 1 × Phosphatase inhibitor (Roche) and treated with 50 μg/mL DNase I and RNase A. Urea-soluble proteins were quantified by Bradford assay. Thirty micrograms total protein from each sample was reduced in 4 mM DTT for 1 h at 20°C, alkylated with 8 mM iodoacetamide for 1 h at 20°C in the dark. Enzymatic digestion was performed first with Lys C (Wako) for 4 h at 37°C (1:75), then with trypsin for 16 h at 37°C (1:75).
Digested peptides were next prefractionated on reverse-phase C18 stage tips in ammonium formate at pH 10. Each peptide fraction was then dried by vacuum centrifugation, reconstituted in 10% formic acid, and subjected to second dimension liquid chromatography-mass spectrometer at low pH. Samples were analyzed on an Orbitrap Q Exactive HFX spectrometer (Thermo Scientific) connected to a UHPLC 1290 system (Agilent). Raw files were processed using MaxQuant version 1.5.3.30 and the Andromeda search engine, against the human Uniprot database (161,042 entries, version Nov 2017).