title: A miniaturized chemical proteomic approach for target profiling of clinical kinase inhibitors in tumor biopsies.
abstract: 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.
authors.affiliation: 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.
authors.forename: Ivo
authors.initials: I
authors.lastname: Chamrád
authors.forename: Uwe
authors.initials: U
authors.lastname: Rix
authors.forename: Alexey
authors.initials: A
authors.lastname: Stukalov
authors.forename: Manuela
authors.initials: M
authors.lastname: Gridling
authors.forename: Katja
authors.initials: K
authors.lastname: Parapatics
authors.forename: André C
authors.initials: AC
authors.lastname: Müller
authors.forename: Soner
authors.initials: S
authors.lastname: Altiok
authors.forename: Jacques
authors.initials: J
authors.lastname: Colinge
authors.forename: Giulio
authors.initials: G
authors.lastname: Superti-Furga
authors.forename: Eric B
authors.initials: EB
authors.lastname: Haura
authors.forename: Keiryn L
authors.initials: KL
authors.lastname: Bennett
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry*; D000818:Animals; D001706:Biopsy; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D002846:Chromatography, Affinity; D000069439:Dasatinib; D006801:Humans; D020014:K562 Cells; D008175:Lung Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D058977:Molecular Sequence Annotation; D058990:Molecular Targeted Therapy; D009368:Neoplasm Transplantation; D009570:Nitriles / Q000737:chemistry*; D060066:Protein Interaction Maps; D047428:Protein Kinase Inhibitors / Q000737:chemistry; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification*; D040901:Proteomics; D011743:Pyrimidines / Q000737:chemistry*; D011804:Quinolines / Q000737:chemistry*; D013844:Thiazoles / Q000737:chemistry*
chemical_list: D000814:Aniline Compounds; D009570:Nitriles; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D011804:Quinolines; D013844:Thiazoles; C471992:bosutinib; D011505:Protein-Tyrosine Kinases; D000069439:Dasatinib
references.citation: Proteomics. 2003 Aug;3(8):1454-63
references.pmid: 12923771
references.citation: Bioinformatics. 2009 Apr 15;25(8):1091-3
references.pmid: 19237447
references.citation: Anal Chem. 2000 May 15;72(10):2247-55
references.pmid: 10845370
references.citation: Nucleic Acids Res. 2006 Jan 1;34(Database issue):D535-9
references.pmid: 16381927
references.citation: N Engl J Med. 2012 Mar 8;366(10):883-892
references.pmid: 22397650
references.citation: Clin Cancer Res. 2008 Oct 15;14(20):6456-68
references.pmid: 18927285
references.citation: Blood. 1975 Mar;45(3):321-34
references.pmid: 163658
references.citation: Nucleic Acids Res. 2008 Jan;36(Database issue):D646-50
references.pmid: 17965090
references.citation: J Med Chem. 2004 Dec 30;47(27):6658-61
references.pmid: 15615512
references.citation: Mol Cell Proteomics. 2008 Oct;7(10):1887-901
references.pmid: 18676365
references.citation: Leukemia. 2009 Mar;23(3):477-85
references.pmid: 19039322
references.citation: Blood. 2007 Dec 1;110(12):4055-63
references.pmid: 17720881
references.citation: Nucleic Acids Res. 2007 Jan;35(Database issue):D572-4
references.pmid: 17135203
references.citation: Nat Chem Biol. 2009 Sep;5(9):616-24
references.pmid: 19690537
references.citation: Nat Rev Drug Discov. 2007 Nov;6(11):891-903
references.pmid: 17917669
references.citation: Mol Cell Proteomics. 2009 Dec;8(12):2759-69
references.pmid: 19828875
references.citation: Nucleic Acids Res. 2000 Jan 1;28(1):289-91
references.pmid: 10592249
references.citation: Proc Natl Acad Sci U S A. 2007 Aug 14;104(33):13283-8
references.pmid: 17684099
references.citation: Nat Rev Drug Discov. 2002 Jul;1(7):493-502
references.pmid: 12120256
references.citation: Nat Chem Biol. 2010 Apr;6(4):291-9
references.pmid: 20190765
references.citation: Nucleic Acids Res. 2007 Jan;35(Database issue):D561-5
references.pmid: 17145710
references.citation: Genome Res. 2003 Nov;13(11):2498-504
references.pmid: 14597658
references.citation: Mol Cell Proteomics. 2005 Dec;4(12):2010-21
references.pmid: 16249172
references.citation: Anal Chem. 2010 Mar 15;82(6):2272-81
references.pmid: 20166708
references.citation: Mol Syst Biol. 2008;4:218
references.pmid: 18766178
references.citation: Nat Biotechnol. 2008 Jan;26(1):127-32
references.pmid: 18183025
references.citation: J Proteome Res. 2009 Oct;8(10):4753-65
references.pmid: 19653696
references.citation: Nat Rev Drug Discov. 2010 Jun;9(6):427-8
references.pmid: 20514063
references.citation: Nat Immunol. 2009 Apr;10(4):356-60
references.pmid: 19295632
references.citation: Biochim Biophys Acta. 2009 Apr;1795(2):92-103
references.pmid: 19167460
references.citation: Anal Chem. 2009 Sep 15;81(18):7757-65
references.pmid: 19689114
references.citation: Diagn Pathol. 2007 Aug 23;2:31
references.pmid: 17716363
references.citation: Drug Discov Today. 2009 Nov;14(21-22):1021-9
references.pmid: 19596079
references.citation: Nat Biotechnol. 2011 Oct 30;29(11):1039-45
references.pmid: 22037377
references.citation: J Proteomics. 2011 Feb 1;74(2):151-66
references.pmid: 20940065
references.citation: Nat Rev Drug Discov. 2008 May;7(5):391-7
references.pmid: 18404149
references.citation: Trends Pharmacol Sci. 2010 Feb;31(2):82-8
references.pmid: 20004028