Shotgun proteomics was used to study the steady phosphorylation state of complex I (NADH:ubiquinone oxidoreductase) subunits from bovine heart mitochondria. A total tryptic digestion of enzymatically active complex I was performed and the resulting peptide mixture was subjected to phosphopeptide-enrichment by the use of titanium dioxide (TiO2). The phosphopeptide-enriched fraction was separated and analyzed with nano-scale reverse-phase HPLC-ESI-MS/MS in single information dependent acquisition mode. Hence two phosphorylated complex I subunits were detected: 42 kDa and B14.5a. Phosphorylation of 42 kDa subunit at Ser-59 has already been determined with fluorescent phosphoprotein specific gel staining and mass spectrometry (Schilling, et al. (2005) FEBS Lett. 579, 2485-2490). In our work, this finding has been confirmed using a non gel-based approach. In addition, we report novel phosphorylation on B14.5a nuclear encoded subunit. We demonstrated evidence of the phosphorylation site at Ser-95 residue by collision-induced dissociation experiments on three different molecular ions of two tryptic phosphopeptides of B14.5a.