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Quantitative analysis of design substrates

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Federico Uliana

LAB HEAD: Ruedi Aebersold

PROVIDER: PXD020321 | Pride | 2021-02-04

REPOSITORIES: Pride

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Project description:This model is from the article: A model for the stoichiometric regulation of blood coagulation. Hockin MF, Jones KC, Everse SJ, Mann KG. Journal of Biological ChemistryVolume 277, Issue 21, 24 May 2002, Pages 18322 -18333 11893748, Abstract: We have developed a model of the extrinsic blood coagulation system that includes the stoichiometric anticoagulants. The model accounts for the formation, expression, and propagation of the vitamin K-dependent procoagulant complexes and extends our previous model by including: (a) the tissue factor pathway inhibitor (TFPI)-mediated inactivation of tissue factor (TF).VIIa and its product complexes; (b) the antithrombin-III (AT-III)-mediated inactivation of IIa, mIIa, factor VIIa, factor IXa, and factor Xa; (c) the initial activation of factor V and factor VIII by thrombin generated by factor Xa-membrane; (d) factor VIIIa dissociation/activity loss; (e) the binding competition and kinetic activation steps that exist between TF and factors VII and VIIa; and (f) the activation of factor VII by IIa, factor Xa, and factor IXa. These additions to our earlier model generate a model consisting of 34 differential equations with 42 rate constants that together describe the 27 independent equilibrium expressions, which describe the fates of 34 species. Simulations are initiated by "exposing" picomolar concentrations of TF to an electronic milieu consisting of factors II, IX, X, VII, VIIa, V, and VIIII, and the anticoagulants TFPI and AT-III at concentrations found in normal plasma or associated with coagulation pathology. The reaction followed in terms of thrombin generation, proceeds through phases that can be operationally defined as initiation, propagation, and termination. The generation of thrombin displays a nonlinear dependence upon TF, AT-III, and TFPI and the combination of these latter inhibitors displays kinetic thresholds. At subthreshold TF, thrombin production/expression is suppressed by the combination of TFPI and AT-III; for concentrations above the TF threshold, the bolus of thrombin produced is quantitatively equivalent. A comparison of the model with empirical laboratory data illustrates that most experimentally observable parameters are captured, and the pathology that results in enhanced or deficient thrombin generation is accurately described.

			Action	DRS
	PeptideRTResults.csv	Csv
	analysis.sky	Other
	analysis.sky.view	Other
	analysis.skyd	Other
	vmatej_M1909_118.raw	Raw

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Quantitative analysis of design substrates

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