A Caged Electrophilic Probe for Global Analysis of Cysteine Reactivity in Living Cells

Cysteine residues are subject to diverse modifications, such as oxidation, nitrosation, and lipidation. The resulting loss in cysteine reactivity can be measured using electrophilic chemical probes, which importantly provide the stoichiometry of modification. An iodoacetamide (IA)-based chemical pro...

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Bibliographic Details
Published inJournal of the American Chemical Society Vol. 137; no. 22; pp. 7087 - 7090
Main Authors Abo, Masahiro, Weerapana, Eranthie
Format Journal Article
LanguageEnglish
Published United States American Chemical Society 10.06.2015
Subjects
Cell Line
chelation
cysteine
Cysteine - chemistry
cytotoxicity
disulfide bonds
epidermal growth factor
Humans
irradiation
Ketones - chemistry
Molecular Probes
oxidation
reactive oxygen species
stoichiometry
sulfides
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Summary:Cysteine residues are subject to diverse modifications, such as oxidation, nitrosation, and lipidation. The resulting loss in cysteine reactivity can be measured using electrophilic chemical probes, which importantly provide the stoichiometry of modification. An iodoacetamide (IA)-based chemical probe has been used to concurrently quantify reactivity changes in hundreds of cysteines within cell lysates. However, the cytotoxicity of the IA group precludes efficient live-cell labeling, which is important for preserving transient cysteine modifications. To overcome this limitation, a caged bromomethyl ketone (BK) electrophile was developed, which shows minimal cytotoxicity and provides spatial and temporal control of electrophile activation through irradiation. The caged-BK probe was utilized to monitor cysteine reactivity changes in A431 cells upon epidermal growth factor (EGF)-stimulated release of cellular reactive oxygen species. Decreased reactivity was observed for cysteines known to form sulfenic acids and redox-active disulfides. Importantly, the caged-BK platform provided the first quantification of intracellular disulfide bond formation upon EGF stimulation. In summary, the caged-BK probe is a powerful tool to identify reactivity changes associated with diverse cysteine modifications, including oxidation, metal chelation, and inhibitor binding, within a physiologically relevant context.
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ISSN:0002-7863
1520-5126
1520-5126
DOI:10.1021/jacs.5b04350