PXD000681

PXD000681 is an original dataset announced via ProteomeXchange.

Title	Probing tyrosine kinase inhibitors specificity
Description	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.
HostingRepository	PRIDE
AnnounceDate	2014-07-24
AnnouncementXML	Submission_2014-07-24_03:48:52.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Piero Giansanti
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; phosphorylated residue; iodoacetamide derivatized residue
Instrument	LTQ Orbitrap; LCQ Classic; LTQ Orbitrap Velos; LTQ Orbitrap Elite; TripleTOF 5600

Revision	Datetime	Status	ChangeLog Entry
0	2014-01-13 02:11:18	ID requested
1	2014-05-12 03:20:53	announced
⏵ 2	2014-07-24 03:48:53	announced	Updated project metadata.

Giansanti P, Preisinger C, Huber KV, Gridling M, Superti-Furga G, Bennett KL, Heck AJ, Evaluating the promiscuous nature of tyrosine kinase inhibitors assessed in A431 epidermoid carcinoma cells by both chemical- and phosphoproteomics. ACS Chem Biol, 9(7):1490-8(2014) [pubmed]

ProteomeXchange project tag: PRIME-XS Project

curator keyword: Biomedical

submitter keyword: Human, LC-MS/MS, tyrosine kinase inhibitors, phosphoproteomics, chemical proteomics, phosphotyrosine

Albert J.R. Heck
contact affiliation	Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands. Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, the Netherlands
contact email	a.j.r.heck@uu.nl
lab head
Piero Giansanti
contact affiliation	NPC
contact email	p.giansanti@uu.nl
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2014/05/PXD000681

PRIDE project URI

• PRIDE
  1. PXD000681
     1. Label: PRIDE project
     2. Name: Probing tyrosine kinase inhibitors specificity