Orbitrap qe hf x mass spectrometer

The analysis was performed using a shotgun proteomics approach based on liquid chromatography‐high resolution tandem mass spectrometry. After reduction and alkylation, liver proteins were digested with trypsin (Promega, Madison, Wisconsin, USA) overnight at 37°C, and then, the peptides were collected. Peptide mixtures from each sample were labelled using 16‐plex tandem mass tags (TMT) pro reagents (Thermo Scientific, MA, USA) to quantify up to 16 samples simultaneously in the same analysis. Eight isobaric compounds were used to label different samples of the control group (reporter ions at 126, 127N, 127C, 128N, 128C, 129N, 129C, and 130N), while other tags were used to label the cachexia group sample (reporter ion at 130C, 131N, 131C, 132N, 132C, 133N, 133C, and 134), according to the manufacturer's instructions. A high pH reversed‐phase peptide fractionation was used to fractionate TMT‐labelled peptides by increasing acetonitrile step‐gradient elution. Mass spectrometric data were collected on an Orbitrap QE HF‐X mass spectrometer coupled to an EASY‐nLC™ 1200 UHPLC system (Thermo Fisher Scientific, USA). Raw data were processed using Proteome Discoverer (v2.4) and searched using the Mascot (Matrix Science, London, UK; version 2.2) engine. Detailed MS procedure is provided in Data S1.