Acid elution and one-dimensional shotgun analysis on an Orbitrap mass spectrometer: an application to drug affinity chromatography.
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 microm 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.
Fernbach
Nora V
NV
Center for Molecular Medicine of the Austrian Academy of Sciences (CeMM), Lazarettgasse 19, 1090 Vienna, Austria.
Planyavsky
Melanie
M
Müller
André
A
Breitwieser
Florian P
FP
Colinge
Jacques
J
Rix
Uwe
U
Bennett
Keiryn L
KL
D000144:Acids, Acyclic / Q000737:chemistry; D000814:Aniline Compounds / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D006851:Hydrochloric Acid / Q000737:chemistry; D015151:Immunoblotting; D020014:K562 Cells; D013058:Mass Spectrometry / Q000379:methods*; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D011804:Quinolines / Q000737:chemistry / Q000378:metabolism; D016622:Silver Staining
D000144:Acids, Acyclic; D000814:Aniline Compounds; D009570:Nitriles; D011506:Proteins; D020543:Proteome; D011804:Quinolines; C471992:bosutinib; D010770:Phosphotransferases; D006851:Hydrochloric Acid