Dataset Information

Probing tyrosine kinase inhibitors specificity

ABSTRACT: Using a three-pronged MS-based proteomic approach, we assessed the mode-of-action of four clinically-relevant tyrosine kinase inhibitors in an epithelial cancer cell line. Here we defined the: (I) semi-quantitative tyrosine kinome; (II) drug interactome; and (III) drug-induced effects on the tyrosine signalome. We first examined the entire proteome and kinome profile expressed in the A431 cell line, and determined the relative abundance of all possible protein targets of the tyrosine kinase inhibitors. We therefore utilized a ‘shot-gun’ approach that involved a multidimensional separation-based proteomic strategy. To ensure maximal protein identification, we reduced sample complexity by strong cation exchange (SCX) chromatography, and analyzed all fractions by LC-MS/MS. To determine the direct targets of the four selected TKIs, we subjected the proteins extracted from the A431 cancer cell line to drug-coupled affinity matrices. By comparing the pull-down results from a lysate with a second identical lysate to which 20 uM of the inhibitor had been added to block binding to the beads, we were able to identify selective binding interactors using LC-MS/MS. This differential affinity pull-downs approach allowed us to also quantitatively evaluate the binding affinity of the tyrosine kinases to each of the drugs. To evaluate the global tyrosine phosphorylation dynamics that occurs in A431 cells upon treatment with each of the four inhibitors, phosphotyrosine peptides were enriched and identified by quantitative mass spectrometry. For this purpose, we treated the cells with each inhibitor for 2 h at appropriate concentrations adopted from reported studies. Cells were lysed, and the proteins digested with the proteases Lys-C and trypsin. To allow quantitation of the phosphorylated peptides originating from the three different peptide pools (control/imatinib/dasatinib or control/bosutinib/nilotinib), we used stable isotope dimethyl labeling. Subsequently, tyrosine-phosphorylated peptides were enriched by immunoprecipitation and analyzed by LC-MS/MS.

INSTRUMENT(S): LTQ Orbitrap, LTQ Orbitrap Velos, TripleTOF 5600, LCQ Classic, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Squamous Cell Carcinoma

SUBMITTER: Piero Giansanti

LAB HEAD: Albert J.R. Heck

PROVIDER: PXD000681 | Pride | 2014-05-12

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	M418-F01-HS-P4763-1.dat	Other
	M418-F01-HS-P4763-1.mgf	Mgf
	M418-F01-HS-P4763-1.pidres.xml	Xml
	M418-F01-HS-P4763-1.raw	Raw
	M418-F01-HS-P4763-2.dat	Other

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Publications

Evaluating the promiscuous nature of tyrosine kinase inhibitors assessed in A431 epidermoid carcinoma cells by both chemical- and phosphoproteomics.

Giansanti Piero P Preisinger Christian C Huber Kilian V M KV Gridling Manuela M Superti-Furga Giulio G Bennett Keiryn L KL Heck Albert J R AJ

ACS chemical biology 20140514 7

Deregulation of protein tyrosine kinase signaling has been linked to many diseases, most notably cancer. As a consequence, small molecule inhibitors of protein tyrosine kinases may provide powerful strategies for treatment. Following the successful introduction of imatinib in the treatment of chronic myelogenous leukemia, such drugs are also now evaluated for other types of cancer. However, many developed kinase inhibitors are not very target-specific and therefore may induce side effects. The i ...[more]

PMID: 24804581

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