PXD036024

PXD036024 is an original dataset announced via ProteomeXchange.

Title	Native holdup (nHU) to measure binding affinities from cell extracts
Description	Characterizing macromolecular interactions is essential for understanding cellular processes, yet nearly all methods used to detect protein interactions from cells are qualitative. Here, we introduce the native holdup (nHU) approach to quantify equilibrium binding constants and explore binding mechanisms of protein interactions from cell extracts. Compared to other pulldown-based assays, nHU requires less sample preparation and can be coupled to any analytical methods, such as western blotting (nHU-WB) or mass spectrometry (nHU-MS) as readouts. We use nHU to explore interactions of SNX27, a cargo adaptor of the retromer complex and find good agreement between in vitro affinities and those measured directly from cell extracts using nHU. This challenges the unwritten paradigm stating that biophysical parameters like binding constants cannot be accurately determined from cells or cellular extracts. We discuss the strengths and limitations of nHU and provide simple protocols that can be implemented in most laboratories.
HostingRepository	PRIDE
AnnounceDate	2023-11-14
AnnouncementXML	Submission_2023-11-14_08:41:08.191.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Luc Negroni
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Exploris 480; LTQ Orbitrap Elite

Revision	Datetime	Status	ChangeLog Entry
0	2022-08-12 13:19:26	ID requested
1	2023-03-11 02:56:43	announced
⏵ 2	2023-11-14 08:41:10	announced	2023-11-14: Updated project metadata.

Zambo B, Morlet B, Negroni L, Trave G, Gogl G, Native holdup (nHU) to measure binding affinities from cell extracts. Sci Adv, 8(51):eade3828(2022) [pubmed]

submitter keyword: mass spectrometry, protein-protein interaction, affinity constant determination

Gergo Gogl
contact affiliation	Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U1258/CNRS UMR 7104/Université de Strasbourg, 1 rue Laurent Fries, BP 10142, F-67404 Illkirch, France
contact email	goglg@igbmc.fr
lab head
Luc Negroni
contact affiliation	CNRS
contact email	luc.negroni@igbmc.fr
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/2023/03/PXD036024

PRIDE project URI

• PRIDE
  1. PXD036024
     1. Label: PRIDE project
     2. Name: Native holdup (nHU) to measure binding affinities from cell extracts