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