RedoxiFluor: A microplate technique to quantify target-specific protein thiol redox state in relative percentage and molar terms.

Unravelling how reactive oxygen species regulate fundamental biological processes is hampered by the lack of an accessible microplate technique to quantify target-specific protein thiol redox state in percentages and moles. To meet this unmet need, we present RedoxiFluor. RedoxiFluor uses two spectrally distinct thiol-reactive fluorescent conjugated reporters, a capture antibody, detector antibody and a standard curve to quantify target-specific protein thiol redox state in relative percentage and molar terms. RedoxiFluor can operate in global mode to assess the redox state of the bulk thiol proteome and can simultaneously assess the redox state of multiple targets in array mode. Extensive proof-of-principle experiments robustly validate the assay principle and the value of each RedoxiFluor mode in diverse biological contexts. In particular, array mode RedoxiFluor shows that the response of redox-regulated phosphatases to lipopolysaccharide (LPS) differs in human monocytes. Specifically, LPS increased PP2A-, SHP1-, PTP1B-, and CD45-specific reversible thiol oxidation without changing the redox state of calcineurin, PTEN, and SHP2. The relative percentage and molar terms are interpretationally useful and define the most complete and extensive microplate redox analysis achieved to date. RedoxiFluor is a new antibody technology with the power to quantify relative target-specific protein thiol redox state in percentages and moles relative to the bulk thiol proteome and selected other targets in a widely accessible, simple and easily implementable microplate format.