Proteomics of BRAF-inhibitor Resistant Cells

Proteomics was performed as essentially as previously described^{26 (link)}. Briefly, parental or chronic treated BRAF inhibitor resistant K029A cells (1 × 10⁷ cells) were treated with pitavastatin (1 µM; 24 h), and subsequently trypsin treated and collected by centrifugation. Cell pellets were solubilized in the lysis buffer (8 M Urea/100 mM HEPES pH8.5) reduced with 5 mM TCEP at 60 °C for 30 min, alkylated with 14 mM iodoacetamide for 45 min in dark, and precipitated using methanol chloroform at the ratio as (sample: methanol: chloroform: H₂O; 1: 3: 1: 2.5). Samples were centrifuged (4000 g; 10 min) and pellets were washed three times with cold methanol. Extracted proteins were resuspended in 200 mM EPPS buffer and digested with Lys-C (1:100 protease-to-protein ratio) (3 h; 37 °C) following trypsin digestion (1:100 protease-to-protein ratio) at 37 °C overnight. Peptides were quantified by microBCA reagent (Thermo Fisher Scientific) and TMT labeled (6–8 M excess). Formic acid was added to a final concentration of 1% and peptides were clean up using a 50 mg Seppak column. Eluted peptides were dried down and resuspended in 5% ACN/5% formic acid buffer. Ratios were checked by HPLC, and samples fractionated. Fractions were resuspended in 1% formic acid and cleaned up using C18-membrane stage tips. Samples were dried down and MS analysis was performed as previously described.