Quantification of the Redox State of Integrin Disulfide Bonds.

We have developed a mass spectrometry-based method to quantify the redox state of protein disulfide bonds using a pair of isotopic alkylators. This technique can be applied in several contexts to investigate disulfide bond regulation in integrins. It may be used to identify disulfide-bond targets of vascular thiol isomerases, such as the two disulfide bonds in the β2 subunit of the integrin Mac-1 that are modulated by protein disulfide isomerase (PDI), or to evaluate the redox state of wild-type and mutant αIIbβ3 integrins recombinantly expressed in mammalian cells. Additionally, the method enables direct assessment of disulfide bond redox states in platelet integrins under native conditions. These streamlined protocols eliminate the immunoprecipitation steps included in previous workflows, significantly reducing both the time and cost of sample preparation and removing the dependency on high-affinity antibodies. In particular, the exclusion of immunoprecipitation in the platelet protocol ensures that no selection bias is introduced into the protein pool, allowing for a more accurate quantification of endogenous disulfide redox states in platelet proteins.