SILAC-based quantitative MS analysis of proteome labeled by covalent hDM2 inhibitor

SJSA1 cells grown in isotopically "light" or "heavy" medium were treated with Nutlin-3 (10 µM, 4 h). After removal of Nutlin-3 by washing, "light" amino acid-labeled cells were treated with DMSO or covalent hDM2 inhibitor (1 µM) in the presence of Nutlin-3 (10 µM) for 1 h, whereas "heavy" amino acid-labeled cells were treated with covalent hDM2 inhibitor (1 µM). Each cell population were lysed and combined in a 1:1 ratio. The covalent hDM2 inhibitor-modified proteins were conjugated to azide-PEG3-desthiobiotin and purified with Neutravidin beads. Enriched proteins were then subjected to in-gel trypsinization and the resultant peptides were analyzed by LC-MS/MS.