PXD005422

PXD005422 is an original dataset announced via ProteomeXchange.

Title	Methylated-antibody affinity purification to improve proteomic identification of plant RNA polymerase Pol V complex and the interacting proteins
Description	Affinity purification followed by enzymatic digestion and mass spectrometry has been widely utilized for the sensitive detection of interacting proteins and protein complexes in various organisms. In plants, the method is technically challenging due to the low abundance proteins, non-specific binding and difficulties of eluting interacting proteins from antibody beads. In this report, we describe a strategy to modify antibodies by reductive methylation of lysines without affecting their binding properties, followed by on-bead digestion of bound proteins with endoproteinase Lys-C. By this method, the antibody remains intact and does not interfere with the downstream identification of interacting proteins. Non-specific binding proteins were excluded using 14N/15N-metabolic labeling of wild-type and the transgenic plant counterparts. The method was employed to identify 12 co-immunoprecipitated protein subunits in Pol V complex and to discover 17 potential interacting protein targets in Arabidopsis. Our results demonstrated that the modification of antibodies by reductive dimethylation can improve the reliability and sensitivity of identifying low-abundance proteins through on-bead digestion and mass spectrometry. We also show that coupling this technique with chemical crosslinking enables in-depth characterization of endogenous protein complexes and the protein-protein interaction networks including mapping the surface topology and post-translational modifications of interacting proteins.
HostingRepository	PRIDE
AnnounceDate	2017-02-28
AnnouncementXML	Submission_2017-02-28_05:23:13.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Guochen Qin
SpeciesList	scientific name: Arabidopsis thaliana (Mouse-ear cress); NCBI TaxID: 3702;
ModificationList	monohydroxylated residue; deamidated residue
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2016-11-22 06:27:00	ID requested
⏵ 1	2017-02-28 05:23:14	announced

Qin G, Ma J, Chen X, Chu Z, She YM, Methylated-antibody affinity purification to improve proteomic identification of plant RNA polymerase Pol V complex and the interacting proteins. Sci Rep, 7():42943(2017) [pubmed]

curator keyword: Technical

submitter keyword: AP-MS, reductive methylation, Pol V complex, chemical cross-link, on-bead digestion

Yi-Min She
contact affiliation	Shanghai center for plant stress biology, Shanghai institutes for biological sciences, Chinese academy of sciences.
contact email	yiminshe@gmail.com
lab head
Guochen Qin
contact affiliation	Chinese academy of sciences
contact email	guochenfirst@sina.com
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/2017/02/PXD005422

PRIDE project URI

• PRIDE
  1. PXD005422
     1. Label: PRIDE project
     2. Name: Methylated-antibody affinity purification to improve proteomic identification of plant RNA polymerase Pol V complex and the interacting proteins