Chemical Proteomics Methods and Protocols
The multidisciplinary science of chemical proteomics studies how small molecules of synthetic or natural origin bind to proteins and modulate their function. In Chemical Proteomics: Methods and Protocols, expert researchers in the field provide key
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Introduction Experimental procedures commonly employed in chemical proteomics approaches can be categorized into two major groups (Fig. 1): (1) global proteomics approaches, aiming at the cell-wide characterization of cellular response to drug treatment, e.g., altered protein expression levels and posttranslational modifications; and (2) activity- or affinity-based protein profiling. The latter summarizing targeted chemoproteomic approaches which employ small molecular probes engineered to selectively capture protein targets/ subproteomes via a specific binding mode (e.g., by targeting cofactor binding sites) (1–8).
Gerard Drewes and Marcus Bantscheff (eds.), Chemical Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 803, DOI 10.1007/978-1-61779-364-6_1, © Springer Science+Business Media, LLC 2012
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Fig. 1. Experimental workflows in chemical proteomics. (a) Global proteomics approaches: Cells are treated with a compound, proteins are extracted from the sample, digested to peptides and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). (b) Activity-based protein profiling (ABPP) utilizes reactive probes specifically targeting the active site of an enzyme family. After protein extraction, the lysate is incubated with the probe to covalently attach to its targets. In the second step, probes and targets are purified using affinity chromatography before digestion and LC-MS/MS analysis. Pretreatment of cells with a small molecule compound binding to the active site of the investigated enzyme family leads to reduced capturing of the target enzyme via the reactive probes. (c) Alternatively, the compounds of interest can be modified and immobilized on a solid support. The immobilized drug is subsequently incubated with a cell extract to specifically enrich for target proteins that are subsequently identified by mass spectrometry. Competition with free excess inhibitor reduces the abundance of captured target proteins.
2. Global Chemoproteomic Profiling
For global chemoproteomic profiling, cells or animals are treated with a drug before system-wide proteome analysis to evaluate the cellular response in a global way (9–12). This strategy has become very attractive because of its simplicity and the unbiased nature of the analysis. Apart from cell permeability, there are no particular requirements for the small molecule compounds to be tested in such assays and the required quantitative mass spectrometric techniques are now available in many laboratories (for recent reviews, see refs. 13–16). However, since typically no protein enrichment is used, the changes that can be observed are limited by the analytical depth of the
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