Dataset Information

PeptideShaker - A complex standard for protein identification, designed by evolution

ABSTRACT: Mass spectrometry-based proteomics data, obtained experimentally or via public repositories, remains challenging to analyze. This challenge is particularly felt when re-analysing or re-purposing public data sets, endeavours that otherwise hold great potential. PeptideShaker is an open source system that greatly simplifies the interpretation and dissemination of proteomics data, and that automates the re-analysis of public data, thus completing the proteomics data cycle (http://peptide-shaker.googlecode.com).

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Pyrococcus Furiosus

TISSUE(S): Whole Body

SUBMITTER: Marc Vaudel

LAB HEAD: Lennart Martens

PROVIDER: PXD001077 | Pride | 2014-06-24

REPOSITORIES: Pride

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A complex standard for protein identification, designed by evolution.

Vaudel Marc M Burkhart Julia M JM Breiter Daniela D Zahedi René P RP Sickmann Albert A Martens Lennart L

Journal of proteome research 20120913 10

Shotgun proteomic investigations rely on the algorithmic assignment of mass spectra to peptides. The quality of these matches is therefore a cornerstone in the analysis and has been the subject of numerous recent developments. In order to establish the benefits of novel algorithms, they are applied to reference samples of known content. However, these were recently shown to be either too simple to resemble typical real-life samples or as leading to results of lower accuracy as the method itself. ...[more]

PMID: 22489649

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Project 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.

			Action	DRS
	Velos005137.mgf	Mgf
	Velos005137.pride.mgf.gz	Mgf
	Velos005137.raw	Raw
	supp_info.mzid.gz	Mzid
	supp_info.pride.mztab.gz	Mztab