PXD042936-1

PXD042936 is an original dataset announced via ProteomeXchange.

Title	High Efficiency Enrichment by Saturating Nanoliters of Protein Affinity Media
Description	Affinity-purification mass-spectrometry (AP-MS) is an established technique for identifying protein-protein interactions (PPIs). The basic technology involves immobilizing a high-specificity ligand to a solid phase support (e.g., an agarose or magnetic bead) to pull down protein(s) of interest from cell lysates. Although these supports are engineered to minimize interactions with background protein, the conventional method recovers mostly non-specific binders. The law of mass action for dilute solutions has taught us to use an excess of beads to capture all target proteins, especially weakly interacting ones. However, modern microbead technology presents a binding environment that is much different from dilute solution. We describe a fluidic platform that captures and processes ultralow nanoliter quantities of magnetic particles, simultaneously increasing the efficiency of PPI detection and strongly suppressing non-specific binding. We demonstrate the concept with synthetic mixtures of tagged protein and illustrate performance with a variety of AP-MS experiment types. These include a BioID experiment targeting lamin-A interactors from HeLa cells, and pulldowns using GFP-tagged proteins associated with a double-strand DNA repair mechanism. We show that efficient extraction requires saturation of the solid-phase support and that <10 nL of beads is sufficient to generate comprehensive protein interaction maps.
HostingRepository	PRIDE
AnnounceDate	2023-09-05
AnnouncementXML	Submission_2023-09-04_17:30:58.158.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	ShaunakRaval
SpeciesList	scientific name: Cricetulus griseus (Chinese hamster) (Cricetulus barabensis griseus); NCBI TaxID: 10029; scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	biotinylated residue; monohydroxylated residue
Instrument	Orbitrap Eclipse

Revision	Datetime	Status	ChangeLog Entry
0	2023-06-13 01:43:29	ID requested
⏵ 1	2023-09-04 17:30:58	announced

Dataset with its publication pending

submitter keyword: BioID, Magnetic beads, Enrichment efficiency, Affinity Purification

David C.Schriemer
contact affiliation	Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada
contact email	dschriem@ucalgary.ca
lab head
ShaunakRaval
contact affiliation	University of Calgary
contact email	sraval@ucalgary.ca
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/2023/09/PXD042936

PRIDE project URI

• PRIDE
  1. PXD042936
     1. Label: PRIDE project
     2. Name: High Efficiency Enrichment by Saturating Nanoliters of Protein Affinity Media