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Dabcyl/Fluorescein‐based Probes for Detection of Thiols and Disulfides: Proteomic Application to Discovery of Reactive Disulfides in Live Cells

Most diseases produce some perturbation of the levels and redox state of cellular thiols. Herein, we report an improved version of our previously reported fluorescent‐dithio probes for detecting cellular thiols, referred to as PMR‐CYS‐FITC, where PMR is p‐methyl red (aka dabcyl), a fluorescein quencher. Unlike most thiol‐reactive probes, PMR‐CYS‐FITC can also detect certain disulfides, making it useful for ‘disulfide proteomics’. PMR‐CYS‐FITC was used to identify new disulfide‐containing protein(s) in the zebrafish chorion, with potential role(s) in protecting the embryo during development. Confocal microscopy of the embryo, followed by in‐gel staining and imaging of proteins, then tandem MS, led to our discovery of two novel disulfide‐containing proteins in the fish chorion, lipovitellin and C‐reactive protein (CRP). CRP is a well‐known biomarker for infection or inflammation (associated with oxidative stress) in humans, so discovery of CRP in the zebrafish has relevance for its use as a model organism. In vitro biophysical characterization of this probe provides further explanations for the differential reactivity of disulfides, and enhanced fluorescence for some fluorescein‐protein mixed disulfides. Research was supported by BTA grant #01‐KMS LEG FY06‐12368.