PXD000282

PXD000282 is an original dataset announced via ProteomeXchange.

Title	EPAC1 knockout mouse effects in platelets and plasma
Description	ABSTRACT FOR BOTH PLASMA AND PLATELET-LYSATE In this article we describe the involvement of exchange factor activated by cAMP 1 (Epac1) in hemostasis and platelet activation. We have used plasma and platelet-lysate from EPAC1 knockout mice and wild-type control mice in label-free proteomics with an Orbitrap Velos Pro. Blood was obtained from wild-type and Epac1-/- mice euthanized with CO2. Approximately 1000 ul was drawn from the left ventricle into a 2 ml syringe, containing 100 ul ACD and 200 ul modified Tyrode`s buffer. The blood was centrifuged at 200g for 5 minutes at room temperature, and the resulting platelet-rich plasma (PRP) centrifuged at 700g for another 10 minutes in the presence of 10 ul ACD. The resulting platelet-poor plasma (PPP) was transferred to Eppendorf tubes and the pelleted platelets resuspended in modified Tyrode`s buffer and adjusted to 2.5 x 108 platelets/ml. Platelets were allowed to rest at room temperature before experimentation. For proteomics analysis, platelets were further purified by size-exclusion through Sepharose CL-2B gel (Pharmacia Biotec, Sweden) as previously described by Jensen et al. in Blood 2004; 104. Plasma was crude or depleted for Albumin prior to trypsination and LCMS with quantification using Progenesis LCMS. The platelet-lysates were fractioned on SDS-PAGE prior to trypsination and LC-MS analysis with MaxQuant quantification. Label-free protein quantification for plasma The software Progenesis LC-MS® Ver 2.7 (Nonlinear Dynamics Ltd, Newcastle, UK) was used for label-free quantification and comparison of LC-MS proteomics data based on the volume, m/z and retention time of the MS1 features (peptides). In Progenesis, the LC-MS runs were automatically aligned, and only features with charges between +2 to +7 and containing associated MSMS spectra were accepted for export as an mgf file for identification. The mgf file was search against the human SwissProt Mus musculus database (version September 2012) using SearchGUI Ver 1.8.9. The search criteria were: trypsin as the protease with no miss-cleavages accepted, fixed carbamidomethylation on cystein, variable oxidation on methionine, precursor mass tolerance of 10 ppm, fragment mass tolerance of 0.7 and OMSSA as the search engine. The search result and associated spectra were combined and assigned to proteins in Peptide Shaker Ver 0.17.3 (http://peptideshaker.googlecode.com) at 1% FDR. The results were exported from PeptideShaker as validated PSMs in a Phenyx format, and imported back into Progenesis. The protein abundances reported from Progenesis were based on the sum of the normalized abundance of the unique identified petides. The proteomics raw files and PRIDE XML identification files were uploaded to PRIDE using ProteomeXchange ver 1.0.4, and the projects are public available. Two PRIDE XML files were generated using PeptideShaker, one for crude plasma and one for albumin-depleted plasma.
HostingRepository	PRIDE
AnnounceDate	2017-07-25
AnnouncementXML	Submission_2017-08-28_02:41:55.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD000282
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Eystein Oveland
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	monohydroxylated residue; iodoacetamide derivatized amino-terminal residue: 57.021464; acetylated residue; monohydroxylated lysine: 15.994915; iodoacetamide derivatized residue
Instrument	instrument model: LTQ-Orbitrap; instrument model: LTQ Orbitrap; LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2013-06-10 03:48:22	ID requested
1	2017-07-25 08:31:28	announced
⏵ 2	2017-08-28 02:41:57	announced	Updated publication reference for PubMed record(s): 28821815.

Nygaard G, Herfindal L, Asrud KS, Bj, ø, rnstad R, Kopperud RK, Oveland E, Berven FS, Myhren L, Hoivik EA, Lunde THF, Bakke M, D, ø, skeland SO, Selheim F, expression. Sci Rep, 7(1):8725(2017) [pubmed]

curator keyword: Biomedical

submitter keyword: EPAC1 knockout, LC-MS, label-free, platelets, plasma

Professor PhD Frode Selheim
contact affiliation	Proteomics Unit at the University of Bergen, Department of Biomedicine, University of Bergen
contact email	Frode.Selheim@uib.no
lab head
Eystein Oveland
contact affiliation	Proteomics Unit at University of Bergen
contact email	eystein.oveland@biomed.uib.no
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/2017/07/PXD000282

PRIDE project URI

• PRIDE
  1. PXD000282
     1. Label: PRIDE project
     2. Name: EPAC1 knockout mouse effects in platelets and plasma