6 plex tandem mass tag reagents

From each sample, 35 μg of acetone-precipitated proteins were subjected to reduction, alkylation, digestion and labelled using 6-plex Tandem Mass Tag reagents according to manufacturer instructions (Thermo Scientific) with some modification, as described previously [24 (link)]. In short, 35 μg of sample and internal standards were reduced with 200 mM DTT (Sigma-Aldrich), alkylated with 375 mM iodoacetamide (Sigma- Aldrich) and precipitated with ice-cold acetone (VWR, Pennsylvania, USA) overnight. Samples were then centrifuged and acetone was decanted. Pellets were resuspended with 50 μl of 100 mM TEAB buffer and digested with trypsin (Promega) overnight at 37 °C (2.5 μg of trypsin per 100 μg of protein). TMT label reagents were equilibrated to room temperature, resuspended in anhydrous acetonitrile LC–MS grade (Thermo Scientific) and added to each sample. Labelling reaction was incubated for 1 h at room temperature and then quenched by adding 5% hydroxylamine (Thermo Scientific) for 15 min. Samples were then combined at equal amounts and 6 μg of each mixed sample set was placed in a well of a microplate, vacuum-dried and stored at - 20 °C before further LC–MS/MS analysis. The LC − MS/MS analysis was performed on Dionex Ultimate 3000 RSLS nano flow system (Dionex, Camberley, UK) and Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) as described elsewhere [25 (link)].