Eriksson Oskar, Stopa Jack, Furie Bruce
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
Methods Mol Biol. 2019;1967:165-182. doi: 10.1007/978-1-4939-9187-7_10.
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.
蛋白质二硫键异构酶（PDI）由活化的血小板和内皮细胞分泌，是血管损伤时血栓形成所必需的。PDI催化其底物蛋白中二硫键的还原、氧化或异构化。在血栓形成过程中，PDI的特定底物在很大程度上仍然难以捉摸，部分原因是PDI与底物相互作用的短暂性。为了克服这一挑战，我们采用并开发了一种动力学底物捕获策略来鉴定PDI的细胞外底物。通过替换PDI活性位点中的选定氨基酸，我们生成了与底物形成稳定复合物的PDI变体，以便随后进行分离和鉴定。我们在此详细描述底物捕获方法，包括PDI变体的生成和表征、动力学捕获实验以及结合底物的分离和鉴定。该方案可适用于几乎任何生物流体或样品，并可应用于其他细胞外硫醇异构酶。
