Acid Elution and One-Dimensional Shotgun Analysis on an Orbitrap Mass Spectrometer: An Application to Drug Affinity Chromatography

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• 作者：Nora V. Fernbach ; Melanie Planyavsky ; Andr Mller ; Florian P. Breitwieser ; Jacques Colinge ; Uwe Rix ; Keiryn L. Bennett
• 刊名：Journal of Proteome Research
• 出版年：2009
• 出版时间：October 2, 2009
• 年：2009
• 卷：8
• 期：10
• 页码：4753-4765
• 全文大小：238K
• 年卷期：v.8,no.10(October 2, 2009)
• ISSN：1535-3907

文摘

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 μm 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 × 10p>6p> 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.