ALISA: A microplate assay to measure protein thiol redox state

Abstract

Measuring protein thiol redox state is central to understanding redox signalling in health and disease. The lack of a microplate assay to measure target specific protein thiol redox state rate-limits progress on accessibility grounds: redox proteomics is inaccessible to most. Developing a microplate assay is important for accelerating discovery by widening access to protein thiol redox biology. Beyond accessibility, enabling high throughput time- and cost-efficient microplate analysis is important. To meet the pressing need for a microplate assay to measure protein thiol redox state, we present the Antibody-Linked Oxi-State Assay (ALISA). ALISA uses a covalently bound capture antibody to bind a thiol-reactive fluorescent conjugated maleimide (F-MAL) decorated target. The capture antibody-target complex is labelled with an amine-reactive fluorescent N-hydroxysuccinimide ester (F-NHS) to report total protein. The covalent bonds that immobilise the capture antibody to the epoxy group functionalised microplate enable one to selectively elute the target. Target specific redox state is ratiometrically calculated as: F-MAL (i.e., reversible thiol oxidation)/F-NHS (i.e., total protein). After validating the assay principle (i.e., increased target specific reversible thiol oxidation increases the ratio), we used ALISA to determine whether fertilisation-a fundamental biological process-changes Akt, a serine/threonine protein kinase, specific reversible thiol oxidation. Fertilisation significantly decreases Akt specific reversible thiol oxidation in Xenopus laevis 2-cell zygotes compared to unfertilised eggs. ALISA is an accessible microplate assay to advance knowledge of protein thiol redox biology in health and disease.

Keywords: Development; Oxidative stress; Protein thiol; Reactive oxygen species; Redox signalling.