Dataset Information

Standardization of mitochondrial proteomics

ABSTRACT: 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 50mM ammonium bicarbonate. Before digestion the proteins were first quantified according to the Bradford assay and then reduced with 1mM TCEP 50 mM and alkylated wit IAA 20mM. 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: 10.0 ppm; Fragment Mass Error Tolerance: 0.05 Da; Enzyme specificity: Trypsin; Max Missed Cleavages: 2; Non-specific Cleavage: one; Fixed Modifications: Carbamidomethylation (C); Variable Modifications: Oxidation (M), Acetylation (K) Ubiquitin; Deamidation (NQ); Max variable PTM per peptide: 2. The database searched was custom made and contained Uniprot Swiss Prot Human, common contaminants and yeast enolase (20442 entries).

INSTRUMENT(S): Synapt G2-S HDMS, LTQ Orbitrap Velos, maXis 4G, impact, TripleTOF 5600, LTQ Orbitrap XL, Orbitrap Fusion

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Maurizio Ronci

PROVIDER: MSV000081121 | MassIVE | Tue May 30 04:44:00 BST 2017

SECONDARY ACCESSION(S): PXD006623

REPOSITORIES: MassIVE

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Publications

Toward the Standardization of Mitochondrial Proteomics: The Italian Mitochondrial Human Proteome Project Initiative.

Alberio Tiziana T Pieroni Luisa L Ronci Maurizio M Banfi Cristina C Bongarzone Italia I Bottoni Patrizia P Brioschi Maura M Brioschi Maura M Caterino Marianna M Chinello Clizia C Cormio Antonella A Cozzolino Flora F Cunsolo Vincenzo V Fontana Simona S Garavaglia Barbara B Giusti Laura L Greco Viviana V Lucacchini Antonio A Maffioli Elisa E Magni Fulvio F Monteleone Francesca F Monti Maria M Monti Valentina V Musicco Clara C Petrosillo Giuseppe G Porcelli Vito V Saletti Rosaria R Scatena Roberto R Soggiu Alessio A Tedeschi Gabriella G Zilocchi Mara M Roncada Paola P Urbani Andrea A Fasano Mauro M

Journal of proteome research 20170913 12

The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven differen ...[more]

PMID: 28828861

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