PXD050236

PXD050236 is an original dataset announced via ProteomeXchange.

Title	Non-prime- and Prime-side Profiling of Pro-Pro Endopeptidase Specificity Using Synthetic Combinatorial Peptide Libraries and Mass Spectrometry
Description	A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), possess the unique ability to hydrolyze proline-proline bonds in proteins. Since a protease’s function is largely determined by its substrate specificity, methods that can extensively characterize substrate specificity are valuable tools for protease research. Previously, we achieved an in-depth characterization of PPEP prime-side specificity. However, PPEP specificity is also determined by the non-prime-side residues in the substrate. To gain a more complete insight into the determinants of PPEP specificity, we characterized the non-prime- and prime-side specificity of various PPEPs using a combination of synthetic combinatorial peptide libraries and mass spectrometry. With this approach, we deepened our understanding of the P3-P3’ specificities of PPEP-1 and PPEP-2, while identifying PPEP-2’s endogenous substrate as the most optimal substrate in our library data. Furthermore, by employing the library approach, we investigated the altered specificity of mutants of PPEP-1 and PPEP-2. Additionally, we characterized a novel PPEP from Anoxybacillus tepidamans, which we termed PPEP-4. Based on structural comparisons, we hypothesized that PPEP-4 displays a PPEP-1-like prime-side specificity, which was substantiated by the experimental data. Intriguingly, another putative PPEP from Clostridioides difficile, CD1597, did not display Pro-Pro endoproteolytic activity. Collectively, we characterized PPEP specificity in detail using our robust peptide library method and, together with additional structural information, provide more insight into the intricate mechanisms that govern protease specificity.
HostingRepository	PRIDE
AnnounceDate	2025-11-11
AnnouncementXML	Submission_2025-11-11_05:55:58.373.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Bart Claushuis
SpeciesList	scientific name: Anoxybacillus tepidamans; NCBI TaxID: NEWT:265948; scientific name: Peptoclostridium difficile (strain 630) (Clostridium difficile); NCBI TaxID: NEWT:272563; scientific name: Paenibacillus alvei; NCBI TaxID: NEWT:44250;
ModificationList	biotinylated residue
Instrument	Orbitrap Fusion Lumos; Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2024-02-29 04:24:45	ID requested
⏵ 1	2025-11-11 05:55:58	announced

10.1111/FEBS.17160;

submitter keyword: Clostridioides difficile, Paenibacillus alvei, PPEP,Protease, specificity, Pro-Pro endopeptidase, Anoxybacillus tepidamans, LC-MS/MS

Peter A. van Veelen
contact affiliation	Center for Proteomics and Metabolomics, Leiden Cniversity Medical Center, The Netherlands
contact email	p.a.van_veelen@lumc.nl
lab head
Bart Claushuis
contact affiliation	Leiden University Medical Center
contact email	b.claushuis@lumc.nl
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD050236
     1. Label: PRIDE project
     2. Name: Non-prime- and Prime-side Profiling of Pro-Pro Endopeptidase Specificity Using Synthetic Combinatorial Peptide Libraries and Mass Spectrometry