PXD015756

PXD015756 is an original dataset announced via ProteomeXchange.

Title	Development of a method for quantitative analysis of in vivo interactions of pluripotency transcription factors Sox2, Oct4, Nanog by mass spectrometry
Description	Protein-protein proximity of core pluripotency transcription factors plays an important role during cell reprogramming. Pluripotent embryonic stem (ES) cell identity is controlled by a trio of transcription factors: Sox2, Oct4, and Nanog. These proteins often bind to closely localized genomic sites. The precise mode by which Sox2, Oct4, and Nanog interact with DNA is likely to make a crucial contribution to their function. Here, a detailed protocol for in vivo detection and quantitative analysis of protein-protein proximity of Sox2 and Oct4 using Proximity Utilizing Biotinylation (PUB) method based on the use of the BAP/BirA (target/enzyme) system is described. The method includes design and cloning of DNA plasmid construct, transient transfection of HEK293T cells, Western blot analysis of nuclei fraction and LC-MS/MS analysis. Experiments with coexpression of BAP-X+BirA-Y (X, Y=Sox2, Oct4 and GFP as control) revealed strong biotinylation level of target proteins when X and Y were pluripotency transcription factors compared with control when X=GFP. Since mass spectrometry provides both high sensitivity and more accurate quantification of data a modified workflow was used, in which SDS-PAGE step was eliminated and His-tagged BAP-fused proteins from cell lysate were purified in 6M guanidine HCl buffer, washed, propionylated, digested directly on the Ni sepharose beads using trypsin and analysed on Q-TOF Impact II instrument. Using mass spectrometry allows making quantitative estimation of in vivo interaction of BAP-Sox2 and BirA-Oct4 which was demonstrated by measuring ratios of biotinylation levels of BAP fused either with Sox2 or GFP at different biotin pulse times. After vector preparation this protocol can be completed in seven working days.
HostingRepository	PRIDE
AnnounceDate	2023-11-14
AnnouncementXML	Submission_2023-11-14_08:50:41.523.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD015756
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Arman Kulyyassov
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	Propionyl; Biotin; Oxidation
Instrument	maXis

Revision	Datetime	Status	ChangeLog Entry
0	2019-10-09 05:19:35	ID requested
1	2020-01-21 02:49:05	announced
2	2020-01-27 01:30:48	announced	2020-01-27: Updated publication reference for PubMed record(s): 21842862. 2020-01-27: Updated publication reference for DOI(s): https://doi.org/10.3390/biom10010142.
⏵ 3	2023-11-14 08:50:44	announced	2023-11-14: Updated project metadata.

10.6019/PXD015756;

Kulyyassov A, Shoaib M, Pichugin A, Kannouche P, Ramanculov E, Lipinski M, Ogryzko V, PUB-MS: a mass spectrometry-based method to monitor protein-protein proximity in vivo. J Proteome Res, 10(10):4416-27(2011) [pubmed]

submitter keyword: mass spectrometry, Oct4, proximity utilizing biotinylation, transcription factors, Sox2,Pluripotency

Arman Kulyyassov
contact affiliation	National Center for Biotechnology
contact email	akulyyasov@gmail.com
lab head
Arman Kulyyassov
contact affiliation	National Center for Biotechnology
contact email	akulyyasov@gmail.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD015756
     1. Label: PRIDE project
     2. Name: Development of a method for quantitative analysis of in vivo interactions of pluripotency transcription factors Sox2, Oct4, Nanog by mass spectrometry