Identification of mitochondrial proteins with a bottom up approach after organelle enrichment and 1D PAGE separation for 5 different cell lines. The bands were cut and the proteins reduced, alkylated and digested with trypsin according to a conventional protocol. Acquisition were performed on the Orbitrap Fusion Tribrid mass spectrometer in top speed mode with 3 seconds cycles. Raw data were processed using PEAKS Studio 7.5 (Bioinformatics Solutions Inc.) and searched using the PEAKS search engine and the SPIDER peptide mutation and homology search tool against neXtProt (July 2017; 42,151 total entries). Parent Mass Error Tolerance was set to 10.0 ppm and Fragment Mass Error Tolerance to 0.6 Da. Other search parameters were trypsin enzyme specificity, two missed cleavages per peptide, fixed Carbamidomethylation of Cys and variable Oxidation of Met, Deamidation of Gln and Asn (NQ), Phosphorylation of Ser, Thr and Tyr and Acetylation of Lys with two variable PTM per peptide. Non-specific cleavage was allowed to only one end of the peptide. FDR estimation was enabled, and precursor options corrected. To follow the HPP Mass Spectrometry Data Interpretation Guidelines Version 2.1 and in view of our goal of finding missing proteins, we set the FDR threshold on PSMs to 2%, typically resulting in FDR on peptides lower than 5% and we filtered out all the proteins identified with only one peptide, resulting in a FDR at the protein level of 0.0%.