Project PXD001825

PRIDE Assigned Tags:
Biomedical Dataset

Summary

Title

Aquired resistance mechanisms to EGFR-specific kinase inhibitors using chemical proteomics

Description

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.1.4.0.5) 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%

Contact

Heiner Koch, Technische Universitaet Muenchen
Prof. Bernhard Kuster, Chair of Proteomics and Bioanalytics, Technical University of Munich, Germany ( lab head )

Submission Date

19/02/2015

Publication Date

20/05/2015

Instrument

LTQ Orbitrap

Software

Not available

Quantification

TIC

Experiment Type

Shotgun proteomics

Publication

    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