Thermo dionex ultimate 3000 rslcnano system

For proteomic analysis, the colon mucosa tissue samples were lysed and labeled with tandem mass tag (TMT, Thermo Scientific, San Jose, CA, USA) according to the manufacturer’s instructions. The labeled peptide samples were pooled into a new vial and dried using SpeedVac (Thermo Scientific). The following processes, including strong cation exchange fraction and liquid chromatography (LC)-mass spectrometry (MS) and database searching, were performed by Poochon Scientific (Frederick, MD) as described previously^{22 (link)}. Briefly, TMT-multiplex labeled peptide mixture (100 µg protein/each plex) fractionation was performed using an Agilent AdvanceBio Column and Agilent UHPLC 1290 system (Agilent, Santa Clara, CA). LC/MS/MS analysis was performed using a Thermo Scientific Q-Exactive hybrid Quadrupole-Orbitrap Mass Spectrometer and Thermo Dionex UltiMate 3000 RSLCnano System (Thermo Scientific). Raw MS data files were searched against the human protein sequence databases obtained from the NCBI website using Proteome Discoverer 1.4 software (Thermo Scientific) based on the SEQUEST and percolator algorithms. The false positive discovery rate (FDR) was set at 5%. The resulting Proteome Discoverer Report from Poochon Scientific contained all assembled proteins with peptide sequences and peptide spectrum match counts (PSM#) and TMT-tag-based quantification ratios.