title: Drug Target Identification Using an iTRAQ-Based Quantitative Chemical Proteomics Approach-Based on a Target Profiling Study of Andrographolide.
abstract: Identifying the cellular binding targets of drugs and other bioactive small molecules is a crucial step for understanding their molecular mechanisms of action as well as potential off-target effects. The field of chemical proteomics is an emerging discipline in chemical biology using synthetic chemistry and high-throughput detection techniques to study small molecule-protein interactions. In this chapter, we describe a quantitative chemical proteomics protocol combining bioorthogonal click chemistry and quantitation by isobaric tags for relative and absolute quantification (iTRAQ) to identify the specific binding targets of drugs and bioactive small molecules such as natural products. A modified drug probe with a click chemistry-enabling addition is synthesized and used in live cell treatments where it undergoes covalent interactions with its cognate cellular targets. The probes are then ligated to biotin through click chemistry and enriched with avidin beads, followed by iTRAQ labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for protein identification and relative quantitation discriminating specific targets from nonspecific binding proteins. The presented protocol has been used to successfully profile prominent drugs and natural products including andrographolide, aspirin, curcumin, etc., and can be a powerful tool to study the molecular mechanisms of bioactive small molecules.
authors.affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University, Nanjing, China; Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Faculty of Science, National University of Singapore, Singapore; Interdisciplinary Research Group in Infectious Diseases, Singapore-MIT Alliance for Research & Technology (SMART), Singapore. Electronic address: wangjg@nju.edu.cn.
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.affiliation: Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Faculty of Science, National University of Singapore, Singapore.
authors.forename: Y K
authors.initials: YK
authors.lastname: Wong
authors.affiliation: Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Clinical Research Institute, Zhejiang Provincial People's Hospital, Hangzhou, China.
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Faculty of Science, National University of Singapore, Singapore.
authors.forename: Y-M
authors.initials: YM
authors.lastname: Lee
authors.affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University, Nanjing, China. Electronic address: huazc@nju.edu.cn.
authors.forename: Z-C
authors.initials: ZC
authors.lastname: Hua
authors.affiliation: Yong Loo Lin School of Medicine, National University of Singapore, Singapore; NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore. Electronic address: phsshm@nus.edu.sg.
authors.forename: H-M
authors.initials: HM
authors.lastname: Shen
authors.affiliation: Faculty of Science, National University of Singapore, Singapore. Electronic address: dbslinqs@nus.edu.sg.
authors.forename: Q
authors.initials: Q
authors.lastname: Lin
mesh_terms: D000480:Alkynes / Q000737:chemistry*; D002853:Chromatography, Liquid; D057930:Click Chemistry; D004224:Diterpenes / Q000737:chemistry*; D045325:HCT116 Cells; D006801:Humans; D011485:Protein Binding; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry
chemical_list: D000480:Alkynes; D004224:Diterpenes; D020543:Proteome; C030419:andrographolide
keywords: Andrographolide; Chemical biology; Chemical proteomics; Click chemistry; Mass spectrometry; Mechanism of action; Quantitative proteomics; Target identification; iTRAQ