PXD040302

PXD040302 is an original dataset announced via ProteomeXchange.

Title	Control of TurboID-dependent biotinylation intensity in proximity ligation screens
Description	Proximity 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.
HostingRepository	PRIDE
AnnounceDate	2023-11-14
AnnouncementXML	Submission_2023-11-14_09:00:06.260.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Thomas Krüger
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	biotinylated residue; acetylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2023-02-21 02:17:53	ID requested
⏵ 1	2023-11-14 09:00:06	announced

Dataset with its publication pending

submitter keyword: Biotinylation, Proximity ligation screens, Protein-Protein Interactions, Proteomics

Axel A. Brakhage
contact affiliation	Department 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 email	axel.brakhage@leibniz-hki.de
lab head
Thomas Krüger
contact affiliation	Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute
contact email	thomas.krueger@leibniz-hki.de
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD040302
     1. Label: PRIDE project
     2. Name: Control of TurboID-dependent biotinylation intensity in proximity ligation screens