PXD047274-2

PXD047274 is an original dataset announced via ProteomeXchange.

Title	A Photocrosslinking Probe to Capture the Substrates of Caseinolytic Protease P
Description	Protein homeostasis in bacteria is regulated by proteases such as the tetradecameric caseinolytic protease P (ClpP). Although substrates of ClpP have been successfully deciphered in genetically engineered cells, methods which directly trap processed proteins within native cells remain elusive. Here, we introduce an in situ trapping strategy which utilizes trifunctional probes that bind to the active site serine of ClpP and capture adjacent substrates with an attached photocrosslinking moiety. After enrichment using an alkyne handle, substrate deconvolution by mass spectrometry (MS) is performed. We show that our two traps bind substoichiometrically to ClpP, retain protease activity, exhibit unprecedented selectivity for Staphylococcus aureus ClpP in living cells and capture numerous known and novel substrates. The exemplary validation of trapped hits using a targeted proteomics approach confirmed the fidelity of this technology. In conclusion, we provide a novel chemical platform suited for the discovery of serine protease substrates beyond genetic engineering.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_07:00:43.597.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Nina Bach
SpeciesList	scientific name: Staphylococcus aureus; NCBI TaxID: 1280;
ModificationList	acetylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive HF; Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2023-11-27 06:55:20	ID requested
1	2024-10-04 04:50:35	announced
⏵ 2	2024-10-22 07:00:44	announced	2024-10-22: Updated project metadata.

10.1002/anie.202409220;

submitter keyword: S. aureus, substrates

Stephan Sieber
contact affiliation	TUM School of Natural Sciences, Department Biosciences, Chair of Organic Chemistry II, Center for Functional Protein Assemblies (CPA), Technical University Munich (TUM), Ernst-Otto-Fischer Str. 8, Garching, 85748, Germany
contact email	stephan.sieber@tum.de
lab head
Nina Bach
contact affiliation	TU München
contact email	nina.bach@tum.de
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD047274
     1. Label: PRIDE project
     2. Name: A Photocrosslinking Probe to Capture the Substrates of Caseinolytic Protease P