2.2. Sample Preparation for Mass Spectrometry Analysis

CEM T cells were collected and lysed with whole cell lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 1% NP40, 0.1% sodium dodecyl sulphate(SDS)) supplemented with protease inhibitors (Sigma-Aldrich). The insoluble nuclear material was removed by centrifugation at 21,000× g for 20 min. The supernatant was collected and protein concentration was measured using the bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Rockville, MD, USA). Proteins were precipitated with cold acetone. The samples were centrifuged at 13,000× g for 10 min. Pellets were dried for 10 min at room temperature and then re-suspended in 100 mM ammonium bicarbonate buffer containing 10 mM dithiotreitol (DTT). The samples were heated at 95 °C for 5 min to be reduced, then alkylated with 15 mM iodoacetamide and digested with trypsin gold (Thermo Fisher) overnight at 37 °C. Discovery Supelco (Sigma-Aldrich) C18 column was used for peptides separation. Si-propylsulfonic acid (SCX) resin (POROS 50 HS, Perspective Biosystems) column was prepared in a pipette tip and was equilibrated with 0.5% formic acid in 0.25% acetonitrile (equilibration buffer). Samples were eluted by varying concentrations of NaCl solution and dried in a Speed-Vac concentrator centrifuge (Thermo Fisher Scientific).