Identification of PDI Substrates by Mechanism-Based Kinetic Trapping.

Protein disulphide isomerase (PDI) is secreted by activated platelets and endothelial cells and is required for thrombus formation upon vascular injury. PDI catalyzes the reduction, oxidation, or isomerization of disulphide bonds in its substrate proteins. The specific substrates of PDI during thrombus formation have largely remained elusive, in part due to the transient nature of the PDI-substrate interaction.To overcome this challenge we have adapted and developed a kinetic substrate trapping strategy to identify extracellular substrates of PDI. By substitution of selected amino acids in the PDI active sites, we have generated PDI variants that form stable complexes with their substrates for subsequent isolation and identification. We here describe the substrate trapping methodology in detail, including generation and characterization of PDI variants, kinetic trapping experiments, and isolation and identification of bound substrates. The protocol can be adapted for most any biological fluid or sample, and can be applied to other extracellular thiol isomerases.