Proteomics

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Target quantification of octapeptide for Thrombin and FX

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: PXD020320 | Pride | 2021-02-04

REPOSITORIES: Pride

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Publications

Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen.

Uliana Federico F   Vizovišek Matej M   Acquasaliente Laura L   Ciuffa Rodolfo R   Fossati Andrea A   Frommelt Fabian F   Goetze Sandra S   Wollscheid Bernd B   Gstaiger Matthias M   De Filippis Vincenzo V   Auf dem Keller Ulrich U   Aebersold Ruedi R  

Nature communications 20210316 1

Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in  ...[more]

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