Abstract
There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule–protein interactions. We describe a generic approach based on trifunctional Capture Compounds, in which the initial equilibrium-driven interaction between a small molecule probe and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function of the Capture Compound and the surface of the target protein(s). Subsequently, Capture Compound - protein conjugates are isolated from complex biological mixtures via the sorting function of the Capture Compound. Here, we describe the application of a trifunctional Capture Compound that carries the methyltransferase product inhibitor S-Adenosyl-l-homocysteine as the selectivity function for the isolation of methyltransferases from a complex lysate of Escherichia coli DH5α cells. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free S-Adenosyl-l-homocysteine.
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Acknowledgments
This work was supported by the Human Frontier Science Program Organization (HFSP Award 2007, RGP0058/2007-C). We thank Prof. Richard Roberts for initiating the project and for fruitful discussions.
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Lenz, T., Poot, P., Weinhold, E., Dreger, M. (2012). Profiling of Methyltransferases and Other S-Adenosyl-l-Homocysteine-Binding Proteins by Capture Compound Mass Spectrometry. 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_8
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DOI: https://doi.org/10.1007/978-1-61779-364-6_8
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