While targeted therapy based on the idea of attenuating the activity of a preselected, therapeutically relevant protein has become one of the major trends in modern cancer therapy, no truly specific targeted drug has been developed and most clinical agents have displayed a degree of polypharmacology. Therefore, the specificity of anticancer therapeutics has emerged as a highly important but severely underestimated issue. Chemical proteomics is a powerful technique combining postgenomic drug-affinity chromatography with high-end mass spectrometry analysis and bioinformatic data processing to assemble a target profile of a desired therapeutic molecule. Due to high demands on the starting material, however, chemical proteomic studies have been mostly limited to cancer cell lines. Herein, we report a down-scaling of the technique to enable the analysis of very low abundance samples, as those obtained from needle biopsies. By a systematic investigation of several important parameters in pull-downs with the multikinase inhibitor bosutinib, the standard experimental protocol was optimized to 100 μg protein input. At this level, more than 30 well-known targets were detected per single pull-down replicate with high reproducibility. Moreover, as presented by the comprehensive target profile obtained from miniaturized pull-downs with another clinical drug, dasatinib, the optimized protocol seems to be extendable to other drugs of interest. Sixty distinct human and murine targets were finally identified for bosutinib and dasatinib in chemical proteomic experiments utilizing core needle biopsy samples from xenotransplants derived from patient tumor tissue. Altogether, the developed methodology proves robust and generic and holds many promises for the field of personalized health care.

Department of Protein Biochemistry and Proteomics Technological Centre of the Palacký University Centre of the Region Haná for Biotechnological and Agricultural Research Olomouc Czech Republic

Zobrazit více v PubMed

Capdeville R, Buchdunger E, Zimmermann J, Matter A. Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug. Nat. Rev. Drug Discovery. 2002;1:493–502. PubMed

Aggarwal S. Targeted cancer therapies. Nat. Rev. Drug Discovery. 2010;9:427–428. PubMed

Ghoreschi K, Laurence A, O’Shea JJ. Selectivity and therapeutic inhibition of kinases: To be or not to be? Nat. Immunol. 2009;10:356–360. PubMed PMC

Terstappen GC, Schlüpen C, Raggiaschi R, Gaviraghi G. Target deconvolution strategies in drug discovery. Nat. Rev. Drug Discovery. 2007;6:891–903. PubMed

Chan JN, Nislow C, Emili A. Recent advances and method development for drug target identification. Trends Pharmacol. Sci. 2010;31:82–88. PubMed

Goldstein DM, Gray NS, Zarrinkar PP. High-throughput kinase profiling as a platform for drug discovery. Nat. Rev. Drug Discovery. 2008;7:391–397. PubMed

Karaman MW, Herrgard S, Treiber DK, Gallant P, Atteridge CE, Campbell BT, Chan KW, Ciceri P, Davis MI, Edeen PT, Faraoni R, Floyd M, Hunt JP, Lockhart DJ, Milanov ZV, Morrison MJ, Pallares G, Patel HK, Pritchard S, Wodicka LM, Zarrinkar PP. A quantitative analysis of kianse inhibitor selectivity. Nat. Biotechnol. 2008;26:127–132. PubMed

Kruse U, Bantscheff M, Drewes G, Hopf C. Chemical and pathway proteomics. Powerful tools for drug discovery and personalised health care. Mol. Cell. Proteomics. 2008;7.10:1887–1901. PubMed

Bantscheff M, Scholten A, Heck AJR. Revealing promiscuous drug-target interactions by chemical proteomics. Drug Discovery Today. 2009;14:1021–1029. PubMed

Rix U, Superti-Furga G. Target profiling of small molecules by chemical proteomics. Nat. Chem. Biol. 2009;5:616–624. PubMed

van Staveren WC, Solís DY, Hébrant A, Detours V, Dumont JE, Maenhaut C. Human cancer cell lines: Experimental model for cancer in situ? For cancer stem cells? Biochim. Biophys. Acta. 2009;1795:92–103. PubMed

Remsing Rix LL, Rix U, Colinge J, Hantschel O, Bennett KL, Stranzl T, Müller A, Baumgartner C, Valent P, Augustin M, Till JH, Superti-Furga G. Global target profile of the kinase inhibitor bosutinib in primary chronic myeloid leukemia cells. Leukemia. 2009;23:477–485. PubMed

Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey P, Varela I, Phillimore B, Begum S, McDonald NQ, Butler A, Jones D, Raine K, Latimer C, Santos CR, Nohadani M, Eklund AC, Spencer-Dene B, Clark G, Pickering L, Stamp G, Gore M, Szallasi Z, Downward J, Futreal PA, Swanton C. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N. Engl. J. Med. 2012;366:883–892. PubMed PMC

Fernbach N, Planyavsky M, Müller A, Breitweiser FP, Colinge J, Rix U, Bennett KL. Acid elution and one-dimensional shotgun analysis on an Orbitrpmss spectrometer: An application to drug affinity chromatography. J. Proteome Res. 2009;8:4753–4765. PubMed

Rix U, Hantschel O, Dürnberger G, Remsing Rix LL, Planyavsky M, Fernbach NV, Kaupe I, Bennett KL, Valent P, Colinge J, Köcher T, Superti-Furga G. Chemical proteomic profiles of the BCR-ABL inhibitors imatinib, nilotinib, and dasatinib reveal novel kinase and nonkinase targets. Blood. 2007;110:4055–4063. PubMed

Fichtner I, Rolff J, Soong R, Hoffmann J, Hammer S, Sommer A, Becker M, Merk J. Establishment of patient-derived non-small cell lung cancer xenografts as models for the identification of predictive biomarkers. Clin. Cancer Res. 2008;14:6456–6468. PubMed

Bennett KL, Funk M, Tschernutter M, Breitwieser FP, Planyavsky M, Ubaida Mohien C, Müller A, Trajanoski Z, Colinge J, Superti-Furga G, Schmidt-Erfurth U. Proteomic analysis of human cataract aqueous humour: Comparison of one-dimensional gel LCMS with two-dimensional LCMS of unlabelled and iTRAQ-labelled specimens. J. Proteomics. 2011;74:151–166. PubMed

Olsen JV, de Godoy LMF, Li G, Macek B, Mortensen P, Pesch R, Makarov A, Lange O, Horning S, Mann M. Parts per million mass accuracy on an Orbitrpmss spectrometer via lock mass injection into a C-trap. Mol. Cell. Proteomics. 2005;4.12:2010–2021. PubMed

Colinge J, Masselot A, Giron M, Dessingy T, Magnin J. OLAV: Towards high-throughput tandem mass spectrometry data identification. Proteomics. 2003;3:1454–1463. PubMed

Zhang Y, Wen Z, Washburn MP, Florens L. Refinements to label free proteome quantitation: How to deal with peptides shared by multiple proteins. Anal. Chem. 2010;82:2272–2281. PubMed

Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T. Cytoscape: A software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13:2498–2504. PubMed PMC

Bindea G, Mlecnik B, Hackl H, Charoentong P, Tosolini M, Kirilovsky A, Fridman WH, Pages F, Trajanoski Z, Galon J. ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology annotation networks. Bioinformatics. 2009;25:1091–1093. PubMed PMC

Welker L, Akkan R, Holz O, Schultz H, Magnussen H. Diagnostic outcome of two different CT-guided fine needle biopsy procedures. Diagn. Pathol. 2007;2:31. PubMed PMC

Lozzio CB, Lozzio BB. Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood. 1975;45:321–334. PubMed

Anastassiadis T, Deacon SW, Devarajan K, Ma H, Peterson JR. Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat. Biotechnol. 2011;29:1039–1045. PubMed PMC

Kim T, Tolmachev AV, Harkewicz R, Prior DC, Anderson G, Udseth HR, Smith RD. Design and implementation of a new electrodynamic ion funnel. Anal. Chem. 2000;72:2247–2255. PubMed

Second TP, Blethrow JD, Schwartz JC, Merrihew GE, MacCoss MJ, Swaney DL, Russell JD, Coon JJ, Zabrouskov V. Dual-pressure linear ion trpmss spectrometer improving the analysis of complex protein mixtures. Anal. Chem. 2009;81:7757–7765. PubMed PMC

Olsen JV, Schwartz JC, Griep-Raming J, Nielsen ML, Damoc E, Denisov E, Lange O, Remes P, Taylor D, Splendore M, Wouters ER, Senko M, Makarov A, Mann M, Horning S. A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed. Mol. Cell. Proteomics. 2009;8.12:2759–2769. PubMed PMC

Lombardo LJ, Lee FY, Chen P, Norris D, Barrish JC, Behnia K, Castaneda S, Cornelius LA, Das J, Doweyko AM, Fairchild C, Hunt JT, Inigo I, Johnston K, Kamath A, Kan D, Klei H, Marathe P, Pang S, Peterson R, Pitt S, Schieven GL, Schmidt RJ, Tokarski J, Wen ML, Wityak J, Borzilleri RM. Discovery of N-(2-chloro-6-methyl-phenyl)-2-(6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with potent antitumor activity in preclinical assays. J. Med. Chem. 2004;47:6658–6661. PubMed

Hantschel O, Rix U, Schmidt U, Bürckstümmer T, Kneidinger M, Schütze G, Colinge J, Bennett KL, Ellmeier W, Valent P, Superti-Furga G. The Btk tyrosine kinase is a major target of the Bcr-Abl inhibitor dasatinib. Proc. Natl. Acad. Sci. U.S.A. 2007;104:13283–13288. PubMed PMC

Li J, Rix U, Fang B, Bai Y, Edwards A, Colinge J, Bennett KL, Gao J, Song L, Eschrich S, Superti-Furga G, Koomen J, Haura EB. A chemical and phosphoproteomic characterization of dasatinib action in lung cancer. Nat. Chem. Biol. 2010;6:291–299. PubMed PMC

Stark C, Breitkreutz BJ, Reguly T, Boucher L, Breitkreutz A, Tyers M. BioGRID: A general repository for interaction datasets. Nucleic Acids Res. 2006;34:D535–D539. PubMed PMC

Kerrien S, Alam-Faruque Y, Aranda B, Bancarz I, Bridge A, Derow C, Dimmer E, Feuermann M, Friedrichsen A, Huntley R, Kohler C, Khadake J, Leroy C, Liban A, Lieftink C, Montecchi-Palazzi L, Orchard S, Risse J, Robbe K, Roechert B, Thorneycroft D, Zhang Y, Apweiler R, Hermjakob H. IntAct: Open source resource for molecular interaction data. Nucleic Acids Res. 2007;35:D561–D565. PubMed PMC

Chatr-aryamontri A, Ceol A, Palazzi LM, Nardelli G, Schneider MV, Castagnoli L, Cesareni G. MINT: the Molecular INTeraction database. Nucleic Acids Res. 2007;35:D572–D574. PubMed PMC

Lynn DJ, Winsor GL, Chan C, Richard N, Laird MR, Barsky A, Gardy JL, Roche FM, Chan THW, Shah N, Lo R, Naseer M, Que J, Yau M, Acab M, Tulpan D, Whiteside MD, Chikatamarla A, Mah B, Munzner T, Hokamp K, Hancock REW, Brinkman FSL, Innate DB. Facilitating systems-level analyses of the mammalian innate immune response. Mol. Syst. Biol. 2008;4:218. PubMed PMC

Ruepp A, Brauner B, Dunger-Kaltenbach I, Frishman G, Montrone C, Stransky M, Waegele B, Schmidt T, Doudieu ON, Stümpflen V, Mewes HW. CORUM: The comprehensive resource of mammalian protein complexes. Nucleic Acids Res. 2008;36:D646–D650. PubMed PMC

Xenarios I, Rice DW, Salwinski L, Baron MK, Marcotte EM, Eisenberga D. DIP: The Database of Interacting Proteins. Nucleic Acids Res. 2000;28:289–291. PubMed PMC

Chemical proteomic analysis of 6-benzylaminopurine molecular partners in wheat grains