PXD011702

PXD011702 is an original dataset announced via ProteomeXchange.

Title	A well-controlled BioID design for endogenous bait proteins
Description	The CRISPR/Cas9 revolution is profoundly changing the way life sciences technologies are used. Many assays now rely on engineered clonal cell lines to eliminate overexpression issues. Control cell lines are typically provided by non-engineered cells or by engineered clones implying a considerable risk for artefacts because of clonal variation. Genome engineering can also be expected to transform BioID , an elegant proximity labeling method that relies on fusing a bait protein to a promiscuous biotin ligase, BirA*, which results in the tagging of vicinal proteins. We here propose an innovative design to enable BioID for endogenous proteins. To enable this design wherein, we introduced a T2A-BirA* module at the C-terminus of endogenous p53 by genome engineering. This leads to bi-cistronic expression of both p53 and BirA* under control of the endogenous promoter ensuring low background biotinylation in these control cells. By targeting a Cas9-cytidine deaminase base editor to the T2A auto-cleavage site, we can efficiently derive an isogenic population expressing a functional p53-BirA* fusion protein. By A quantitative proteomics workflow we show significant benefits over other experimental settings including forced expression of p53-BirA* and parental control cell lines, and we provide a enabled a ffirst well-controlled view on the proximal proteins of endogenous p53. This novel application for base editors expands the CRISPR/Cas9 toolbox and may prove to be a valuable addition for synthetic biology.
HostingRepository	PRIDE
AnnounceDate	2019-06-17
AnnouncementXML	Submission_2019-06-20_05:29:14.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Giel Vandemoortele
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	biotinylated residue; monohydroxylated residue; acetylated residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2018-11-15 01:14:23	ID requested
1	2019-06-17 07:09:05	announced
⏵ 2	2019-06-20 05:49:33	announced	Updated publication reference for PubMed record(s): 30525648.

Vandemoortele G, De Sutter D, Moliere A, Pauwels J, Gevaert K, Eyckerman S, A Well-Controlled BioID Design for Endogenous Bait Proteins. J Proteome Res, 18(1):95-106(2019) [pubmed]

curator keyword: Technical

submitter keyword: protein protein interaction

BioID proximity labeling

(T2A) autocleavage

genome engineering

p53

base editor

Sven Eyckerman
contact affiliation	1 VIB Center for Medical Biotechnology, VIB, B-9000 Ghent, Belgium 2 Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium
contact email	sven.eyckerman@vib-ugent.be
lab head
Giel Vandemoortele
contact affiliation	VIB-Ugent
contact email	giel.vandemoortele@vib-ugent.be
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/06/PXD011702

PRIDE project URI

• PRIDE
  1. PXD011702
     1. Label: PRIDE project
     2. Name: A well-controlled BioID design for endogenous bait proteins