MassIVE MSV000081329

Complete Public PXD007053

Standardization of mitochondrial proteomics v2 NP

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Description

Comparison of the extraction performances of three different mitochondria enrichment protocols (differential centrifugation, sucrose gradient, a commercial kit based on surfactants) on ten different cell lines. Samples were analysed on several instrumental platforms. Mitochondrial pellets were lysed in RapiGest 0.1% (RG, Waters Corporation) and digested with trypsin in 50 mM ammonium bicarbonate. Before digestion the proteins were first quantified according to the Bradford assay and then reduced with 1 mM TCEP 50 mM and alkylated wit IAA 20 mM. Peptides were recovered by centrifugation and then loaded directly on the respective chromatographic system for the mass spectrometry analysis or on a SDS-PAGE for WB analysis. Raw data were processed with PEAKS 7.5 with the following search parameters. Parent Mass Error Tolerance - depending on the instrument - 10.0 to 40 ppm Fragment Mass Error Tolerance - depending on the instrument - 0.05 to 0.6 Da Enzyme specificity Trypsin Max Missed Cleavages: 2 Non-specific Cleavage: 1 Fixed Modifications: Carbamidomethylation (C) Variable Modifications: Oxidation (M), Deamidation (NQ) Max variable PTM per peptide: 2 The searched database was downloaded from www.nextprot.org and contained the latest annotated human proteome including isoforms (42,151 entries). [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Mt-HPP, Mitochondria enrichment protocol, human cell lines, orbitrap, qtof, high resolution

Contact

Principal Investigators:
(in alphabetical order) 		Maurizio Ronci, University G. d'Annunzio, Italy
Submitting User: cefaclor

Publications

Alberio T, Pieroni L, Ronci M et al.
Toward the Standardization of Mitochondrial Proteomics: The Italian Mitochondrial Human Proteome Project Initiative.
J Proteome Res. 2017 Dec 1;16(12):4319-4329. doi: 10.1021/acs.jproteome.7b00350.

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