The present study describes the free N-terminome analysis of human mitochondria-enriched samples using trimethoxyphenylphosphonium (TMPP) labelling approaches and dN-TOP validation of the proteomics data acquired with an Orbitrap mass spectrometer. The doublet N-terminal oriented proteomics (dN-TOP) strategy is based on a double light/heavy TMPP labelling which enable fast, efficient and reliable high throughput N-terminome data validation