Quantitative analysis of design substrates

Proteases are crucial physiologic regulators of protein structure and function. While proteomic methods contributed extensively to protease characterization efforts, technical challenges remain in terms of throughput, scalability and large datasets of protease cleavages remain scarce. Here, we describe a high-throughput protease screen (HTPS) which allows simultaneous characterization of multiple proteases under various conditions on a microscale 96FASP format. After benchmarking the performance with Trypsin, LysC, GluC, AspN, Chymotrypsin, MMP-2 and MMP-3, we applied it to profile proteases of the blood coagulation cascade (Factors VIIa, IXa, Xa, XIa, Thrombin α, β and γ, Plasmin and Protein C). The large dataset enabled us to map activity, specificity, cleave entropy, allosteric changes of blood cascade proteases induced by Na+ and to predict potential substrates in a data-driven way. Here, we designed two octapeptides, GIPRAAGD (α Thrombin) and GIGRRIAE (Factor Xa), and evaluated their cleavage with the target proteases.