PXD001262

PXD001262 is an original dataset announced via ProteomeXchange.

Title	Affinity capture of S. cerevisiae protein after glutaraldehyde stabilization
Description	It remains extraordinarily challenging to elucidate endogenous protein-protein interactions and proximities within the cellular milieu. The dynamic nature and the large range of affinities of these interactions augment the difficulty of this undertaking. Among the most useful tools for extracting such information are those based on affinity capture of target bait proteins in combination with mass spectrometric readout of the co-isolated species. Although highly enabling, the utility of affinity-based methods is generally limited by difficulties in distinguishing specific from non-specific interactors, preserving and isolating all unique interactions including those that are weak, transient or rapidly exchanging, and differentiating proximal interactions from those that are more distal. Here, we have devised and optimized a set of methods to address these challenges. The resulting pipeline involves flash-freezing cells in liquid nitrogen to preserve the cellular environment at the moment of freezing; cryomilling to fracture the frozen cells into intact sub-micron chunks to allow for rapid access of a chemical reagent and to stabilize the intact endogenous subcellular assemblies and interactors upon thawing; and utilizing the high reactivity of glutaraldehyde to achieve sufficiently rapid stabilization at low temperatures to preserve native cellular interactions. In the course of this work, we determined that relatively low molar ratios of glutaraldehyde to reactive amines within the cellular milieu were sufficient to preserve even labile and transient interactions. This mild treatment enables efficient and rapid affinity capture of the protein assemblies of interest under non-denaturing conditions, followed by bottom-up MS to identify and quantify the protein constituents. For convenience, we have termed this approach Stabilized Affinity Capture Mass Spectrometry (SAC-MS). Here, we demonstrate that SAC-MS allows us to stabilize and elucidate local, distant and transient protein interactions within complex cellular milieux, many of which are not observed in the absence of chemical stabilization.
HostingRepository	PRIDE
AnnounceDate	2018-10-19
AnnouncementXML	Submission_2018-10-19_00:33:56.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Roman Subbotin
SpeciesList	scientific name: Saccharomyces cerevisiae (Baker's yeast); NCBI TaxID: 4932;
ModificationList	No PTMs are included in the dataset
Instrument	LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2014-08-26 01:40:23	ID requested
⏵ 1	2018-10-19 00:33:57	announced

Subbotin RI, Chait BT, A pipeline for determining protein-protein interactions and proximities in the cellular milieu. Mol Cell Proteomics, 13(11):2824-35(2014) [pubmed]

curator keyword: Technical

submitter keyword: budding yeast, glutaraldehyde, affinity capture

Brian T. Chait
contact affiliation	The Rockefeller University
contact email	chait@rockefeller.edu
lab head
Roman Subbotin
contact affiliation	The Rockefeller University
contact email	rsubbotin@rockefeller.edu
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/2018/10/PXD001262

PRIDE project URI

• PRIDE
  1. PXD001262
     1. Label: PRIDE project
     2. Name: Affinity capture of S. cerevisiae protein after glutaraldehyde stabilization