Proteome-Wide Profiling of Targets of Cysteine reactive Small Molecules by Using Ethynyl Benziodoxolone Reagents

• Published in: Angewandte Chemie (International ed.) Vol. 54; no. 37; pp. 10852 - 10857
• Main Authors: Abegg, Daniel, Frei, Reto, Cerato, Luca, Prasad Hari, Durga, Wang, Chao, Waser, Jerome, Adibekian, Alexander
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 07.09.2015; WILEY‐VCH Verlag; Wiley Subscription Services, Inc
• Edition: International ed. in English
• Subjects: activity-based protein profiling; Cellular; Chromatography, Liquid - methods; curcumin; Cysteine; Cysteine - metabolism; Enzymes; HeLa Cells; Humans; Indicators and Reagents - chemistry; Kinases; mass spectrometry; Natural products; Oxolinic Acid - chemistry; Pharmaceutical industry; Profiling; Proteome; Proteomics; Residues; Tandem Mass Spectrometry - methods; target identification; mass spectrometry; activity-based protein profiling; proteomics; target identification; curcumin
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.201505641

In this study, we present a highly efficient method for proteomic profiling of cysteine residues in complex proteomes and in living cells. Our method is based on alkynylation of cysteines in complex proteomes using a “clickable” alkynyl benziodoxolone bearing an azide group. This reaction proceeds fast, under mild physiological conditions, and with a very high degree of chemoselectivity. The formed azide‐capped alkynyl–cysteine adducts are readily detectable by LC‐MS/MS, and can be further functionalized with TAMRA or biotin alkyne via CuAAC. We demonstrate the utility of alkynyl benziodoxolones for chemical proteomics applications by identifying the proteomic targets of curcumin, a diarylheptanoid natural product that was and still is part of multiple human clinical trials as anticancer agent. Our results demonstrate that curcumin covalently modifies several key players of cellular signaling and metabolism, most notably the enzyme casein kinase I gamma. We anticipate that this new method for cysteine profiling will find broad application in chemical proteomics and drug discovery. Good grip: Alkynyl benziodoxolones (EBX reagents) swiftly and highly selectively react with cysteine residues in cellular lysates and in living cells under physiological conditions. A “clickable” EBX probe allowed identification of the biological targets of natural product curcumin. This new method for cysteine labeling is particularly useful for various chemical proteomics applications.