Abstract
The high cost of drug discovery and development requires more efficient approaches to the identification and inhibition of tractable protein targets. One strategy is to pursue families of proteins that already possess affinity for a drug lead scaffold, where that scaffold plays the dual role of serving (a) when tethered to a resin, as a ligand to purify a subproteome of interest, and (b) as a lead molecule that has the potential for optimization for a given member of the subproteome. Here, we describe an example of the purification of a subproteome using a scaffold tailored to the dehydrogenase family of enzymes. Combined with modern LC-MS/MS methods and subsequent searching of proteome databases, such affinity chromatography strategies can be used to purify and identify any proteins with affinity for the scaffold molecule. The method is exemplified using the CRAA (catechol rhodanine acetic acid) privileged scaffold, which is tailored to dehydrogenases. CRAA affinity column chromatography, combined with LC-MS/MS, is described as a method for profiling dehydrogenase subproteomes.
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Acknowledgments
We thank Dr. Bassam Wakim for assistance with mass spectrometry studies. This work was supported in part by GM085739 (NIH) and shared instrumentation grants S10 RR019012 (NIH) and CHE-0521323 (NSF).
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Ge, X., Sem, D.S. (2012). Affinity-Based Profiling of Dehydrogenase Subproteomes. In: Drewes, G., Bantscheff, M. (eds) Chemical Proteomics. Methods in Molecular Biology, vol 803. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-364-6_11
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