Standardization of mitochondrial proteomics v2 NP

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 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).

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: MSV000081329 | MassIVE | Fri Jul 14 07:47:00 BST 2017

SECONDARY ACCESSION(S): PXD007053

REPOSITORIES: MassIVE