Drug Target Identification Using an iTRAQ-Based Quantitative Chemical Proteomics Approach-Based on a Target Profiling Study of Andrographolide

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 chemi...

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Veröffentlicht in:Methods in enzymology 2017, Vol.586, p.291-309
Hauptverfasser: Wang, J, Wong, Y K, Zhang, J, Lee, Y-M, Hua, Z-C, Shen, H-M, Lin, Q
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container_end_page 309
container_issue
container_start_page 291
container_title Methods in enzymology
container_volume 586
creator Wang, J
Wong, Y K
Zhang, J
Lee, Y-M
Hua, Z-C
Shen, H-M
Lin, Q
description 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.
doi_str_mv 10.1016/bs.mie.2016.09.049
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subjects Alkynes - chemistry
Chromatography, Liquid
Click Chemistry
Diterpenes - chemistry
HCT116 Cells
Humans
Protein Binding
Proteome - chemistry
Proteome - isolation & purification
Proteomics
Staining and Labeling
Tandem Mass Spectrometry
title Drug Target Identification Using an iTRAQ-Based Quantitative Chemical Proteomics Approach-Based on a Target Profiling Study of Andrographolide
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