Target Profiling of an Anticancer Drug Curcumin by an In Situ Chemical Proteomics Approach
Interdisciplinary chemical proteomics approaches have been widely applied to the identification of specific targets of bioactive small molecules or drugs. In this chapter, we describe the application of a cell-permeable activity-based curcumin probe (Cur-
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Introduction Chemical proteomics approaches, such as activity-based protein profiling (ABPP), are compound-centric approaches, which combine drug affinity chromatography with mass spectrometry (MS). They have been established as powerful and prevalent tools for the analysis of specific subcellular proteomes [1, 2]. They have been widely used to determinate protein-binding profiles and reveal mechanisms of small-molecule drugs and detect active sites and sites of posttranslational modification of various proteins [3– 6]. ABPP employs synthesized small-molecular probes containing biotin, fluorescent tags such as rhodamine, or clickable tags such as alkyne to covalently attack a distinct class of proteins. Coupled with
Dan-dan Liu, Chang Zou and Jianbin Zhang contributed equally to this work. Glenn R. Hicks and Chunhua Zhang (eds.), Plant Chemical Genomics: Methods and Protocols, Methods in Molecular Biology, vol. 2213, https://doi.org/10.1007/978-1-0716-0954-5_13, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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LC-MS/MS analysis, it has been applied to the detection of interactions of small molecules with proteins, enzyme activity profiling, drug discovery, and target validation [7–9]. Various quantitative proteomics approaches have been established to determine the abundance of proteins. These include the metabolic labeling approaches such as the stable isotope labeling by amino acids in cell culture (SILAC), chemical labeling approaches such as ICAT, tandem mass tag (TMT), bio-orthogonal noncanonical amino acid tagging (BONCAT), and isobaric tag for relative and absolute quantitation (iTRAQ), as well as label-free approaches [10, 11]. Quantitative proteomics approaches have been applied in conjunction with chemical proteomics studies for improved specificity of target identification, such as the profiling of de novo protein synthesis during starvation-mediated autophagy [12, 13], target identification of natural and traditional medicines [10], and exploring the specificity of protein–protein or protein–drug interactions [14–16]. The detailed protocol for target identification that we describe in this chapter is based on in situ proteomic profiling of curcumin targets in HCT116 colon cancer cell line [17]. Briefly, a cellpermeable curcumin probe (Cur-P) with an alkyne moiety, which fully retained the curcumin activity, was synthesized and used to react with the protein targets in live cells, and click chemistry was applied to ligate biotin tags to the protein targets through the alkyne moiety for affinity enrichment and target identification and quantitation, or to ligate a fluorescent dye to the target proteins for visualization in situ. This protocol mainly includes two parts: (1) Unlike the bulky biotin tag, the tiny alkyne group has little effect on the plasma membrane penetration ability of curcumin; thus, the Cur-P can directly target the proteins in vivo. The biotin tag conjugated to the probe by click chemistry after cell lysis. The proteins were then en
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