Proteomics

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Mapping specificity, entropy, allosteric changes and substrates in blood coagulation proteases by a high-throughput protease screen

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.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Matej Vizovisek  

LAB HEAD: Ruedi Aebersold

PROVIDER: PXD018976 | Pride | 2021-02-05

REPOSITORIES: Pride

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Dataset's files

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Action DRS
AspN_results.rar Other
Chy_results.rar Other
FIX_results.rar Other
FVII_results.rar Other
FXI_results.rar Other
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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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