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Mass spectrometric measurement of differential reactivity of cysteine to localize protein-ligand binding sites. Application to tubulin-binding drugs.

作者信息

Kim Yeoun Jin, Pannell Lewis K, Sackett Dan L

机构信息

National Institute of Diabetes, Digestive, Kidney Diseases, National Institutes of Health, U.S. Department of Health and Human Services, Bethesda, MD 20892, USA.

出版信息

Anal Biochem. 2004 Sep 15;332(2):376-83. doi: 10.1016/j.ab.2004.06.033.

Abstract

A new method for localizing binding sites of noncovalent drugs on proteins is presented. We have developed an accurate and high-throughput method based on the mass spectrometric measurement of differential reaction yield of cysteine alkylation (MS-DRC). This method, essentially a semiquantitative footprinting approach, is applicable to any type of ligand targeting cysteine-rich proteins because the method measures the reactivity change of each cysteine toward an alkylating agent instead of monitoring the drug itself. Thus, no modification of the drug is needed. In this study, the method is evaluated using tubulin as a model system. Tubulin and drug-treated tubulin were alkylated separately with several alkylating reagents, followed by proteolysis and high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) and HPLC-MS. Relative alkylation yields of each cysteine toward the reagents were measured by mass spectrometric quantitation. The reaction yields of each cysteine of two samples were compared to detect a particular cysteine (or cysteines) for which reaction yield was markedly decreased following drug binding. Monobromobimane (mBrB) showed the highest differential.Thus, the MS-DRC method with mBrB was evaluated with various tubulin agents, including the covalent agent T138067 and the noncovalent agents colchicine, podophyllotoxin, and 2-methoxyestradiol. Conformational changes induced by drug binding, as well as sites of direct binding, may be identified.

摘要

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