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PXD040302-1

PXD040302 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleControl of TurboID-dependent biotinylation intensity in proximity ligation screens
DescriptionProximity biotinylation screens are a widely used strategy for the unbiased identification of interacting or vicinal proteins. The latest generation biotin ligase TurboID has broadened the range of potential applications, as this ligase promotes an intense and faster biotinylation, even in subcellular compartments like the endoplasmic reticulum. On the other hand, the uncontrollable high basal biotinylation rates deny the system´s inducibility and are often associated with cellular toxicity precluding its use in proteomics. We report here an improved method for TurboID-dependent biotinylation reactions based on the tight control of free biotin levels. Blockage of free biotin with a commercial biotin scavenger reversed the high basal biotinylation and toxicity of TurboID, as shown by pulse-chase experiments. Accordingly, the biotin-blockage protocol restored the biological activity of a bait protein fused to TurboID in the endoplasmic reticulum and rendered the biotinylation reaction inducible by exogenous biotin. Importantly, the biotin-blockage protocol was more effective than biotin removal with immobilised avidin and did not affect the cellular viability of human monocytes over several days. The method presented should be useful to researchers interested in exploiting the full potential of biotinylation screens with TurboID and other high-activity ligases for challenging proteomics questions.
HostingRepositoryPRIDE
AnnounceDate2023-11-14
AnnouncementXMLSubmission_2023-11-14_09:00:06.260.xml
DigitalObjectIdentifier
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterThomas Krüger
SpeciesList scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationListbiotinylated residue; acetylated residue; monohydroxylated residue; iodoacetamide derivatized residue
InstrumentOrbitrap Exploris 480
Dataset History
RevisionDatetimeStatusChangeLog Entry
02023-02-21 02:17:53ID requested
12023-11-14 09:00:06announced
Publication List
Dataset with its publication pending
Keyword List
submitter keyword: Biotinylation, Proximity ligation screens, Protein-Protein Interactions, Proteomics
Contact List
Axel A. Brakhage
contact affiliationDepartment of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute Jena, Leibniz-HKI, Adolf-Reichwein-Str. 23, 07745 Jena, Germany
contact emailaxel.brakhage@leibniz-hki.de
lab head
Thomas Krüger
contact affiliationLeibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute
contact emailthomas.krueger@leibniz-hki.de
dataset submitter
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Dataset FTP location
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