Aquired resistance mechanisms to EGFR-specific kinase inhibitors using chemical proteomics
Here we used kinase affinity enrichment (Kinobeads) and intensity-based mass spectrometry to elucidate aquired resistance mechanisms to EGFR inhibition in cancer cells. First, kinase expression differences between untreated control cells and long term treated resistant cells were investigated using Kinobeads and LC-MS/MS. Overexpressed Kinases were then further investigated by cellular assays to evaluate targets and development of combination treatment strategies.
Sample Processing Protocol
Cells were lysed and lysates were subjected to Kinobeads for affinity enrichment of kinases, Kinases were eluted from kinobeads with 2x NuPAGE LDS buffer containing 50mM DTT and alkylated in 55mM IAA for 30min, Samples were loaded onto a 4-12% Bis-Tris gel for in gel digestion.
Data Processing Protocol
Maxquant (v.18.104.22.168) raw data processing, Uniprot sequence database containing 88,354 sequences, Search parameters: ±20ppm for MS1 first search, ±4.5ppm MS1 main search and 0.5Da MS2 tolerance, Allowed moifications: carbamidometylation (fixed), methionine oxidation and N-terminal protein acetylation (variable), Protein and peptide FDR 1%
Koch H, Busto ME, Kramer K, Médard G, Kuster B. Chemical proteomics uncovers EPHA2 as a mechanism of acquired resistance to small molecule EGFR kinase inhibition. J Proteome Res. 2015 May 12 PubMed: 25963923