10 plex tandem mass tag

Duplicate samples of control and 1.0 μM TMT-treated cell pellets (immature cultures, exposed to the toxicant for 10 d) were solubilized in 6 M urea, 1 mM DTE and 0.1 M TEAB. After homogenization steps, solubilized proteins were recovered by centrifugation (supernatants). Reduction of 10 µg of protein per sample was done with TCEP (tris (2-carboxyethyl)phosphine), final concentration of 10 mM, and samples reacted for 30 min at 30 °C. Alkylation was done with iodoacetamide, added to a final concentration of 40 mM, and samples incubated 60 min in the dark at room temperature. Trypsin was added (ratio of 1:25, w/w), and the digestion was performed overnight at 37 °C. Then, a 10-plex tandem mass tags (Thermo Scientific, Rockford, USA) labeling was performed according to manufacturer’s instructions. Tandem mass tags reagent was dissolved in MeCN, and each sample was incubated 60 min at room temperature with a specific tag. For tandem mass tags quenching, 8 µL of hydroxylamine 5% (v/v) were added and incubated with the samples for 15 min. Tandem mass tags-labeled samples were pooled and dried under vacuum. Samples were dissolved in 5% MeCN/0.1% formic acid and desalted with C18 micro-spin columns (Thermo Scientific). Peptides were separated by off-gel electrophoresis, desalted and solubilized in an appropriate amount of 5% MeCN/0.1% formic acid for mass spectrometry analysis.