Journal
JOURNAL OF PROTEOME RESEARCH
Volume 8, Issue 10, Pages 4753-4765Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr900455x
Keywords
bosutinib; kinase; chemical proteomics; orbitrap; shotgun
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Funding
- Austrian Academy of Sciences
- Austrian Federal Ministry for Science and Research
- Austrian Science Fund FWF
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In this study, the target profile of the promiscuous kinase inhibitor bosutinib from whole cell K562 lysates was investigated by an improved chemical proteomic approach to identify natural binders. By (i) miniaturizing the drug pulldown method, (ii) introducing a 50 mu m inner diameter (i.d.) analytical column for peptide separation, (iii) decreasing the inlet flow rate to 100 nL/minute, and (iv) analyzing the samples on an LTQ Orbitrap XL mass spectrometer, it was clearly demonstrated that the entire approach could be successfully down-scaled by a factor of 100, that is, equivalent to 2 x 10(6) K562 cells. The known major targets of bosutinib were still unequivocally identified in addition to 30 targets not previously identified by gel-based mass spectrometry in our laboratory, In total, 70 individual targets were identified by mass spectrometry across this study of which 19 had not been previously reported. The down-scaled technique was made feasible by eluting the proteins that interact with bosutinib with acid and analyzing the proteins by one-dimensional shotgun proteomics. Overall, these improvements should allow utilization of very limited amounts of patient material to generate a comprehensive and comparative profile of protein drug targets.
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