Aim of the overall project was the definition and characterization of the mitochondrial proteome of human cells, referred to as MitoCoP. To this end, we combined the complementary strengths of subtractive proteomics, ImportOmics and subcellular protein profiling in a global multidimensional classification approach. In this part of the project, we analyzed the correlation between MS analysis time, sample fractionation, use of multiple proteases and replicates on the number of proteins identified and quantified in mitochondrial fractions applying the subtractive proteomics approach.