Rapid and Thiol-Specific High-Throughput Assay for Simultaneous Relative Quantification of Total Thiols, Protein Thiols, and Nonprotein Thiols in Cells

[Image: see text] Thiol groups in biological molecules play a significant role in various physiological functions and pathological conditions. Thiols are divided into two major groups: protein thiols and nonprotein thiols. Numerous methods have been reported for thiol assays. Most of these methods have been developed for glutathione, the principal nonprotein thiol, despite the fact that cellular protein thiols are more abundant than glutathione. Further, these methods usually involve a process of biological sample preparation followed by a separation method, and they are time-consuming. We reported previously a series of thiol-specific fluorogenic benzofurazan sulfides. These nonfluorescent benzofurazan sulfides react rapidly and specifically with a thiol to form a strong fluorescent thiol adduct. The rapid reaction, thiol-specific and fluorogenic nature of the sulfides successfully yielded an application of one of the sulfides for relative quantitation of total thiols in live cells through fluorescence microscopy. In this work, we employed the same compound to develop the first high-throughput method for simultaneous monitoring of protein thiols, nonprotein thiols, and total thiols in cells in a 96-well plate on a fluorescence microplate reader at λ(ex) = 430 nm and λ(em) = 520 nm, respectively. The method is rapid and sensitive, and has been validated by an HPLC thiol assay method. The method can detect thiols with cell concentrations as low as 500 cells/well. We also demonstrated that the method can readily monitor changes in cellular thiol levels. Although the method cannot provide an absolute quantification for thiols because fluorescence intensity of different thiol adducts varies, it provides an accurate measurement of relative quantification, relative to the control. The method will be a valuable tool in thiol-related biomedical/pharmaceutical research.