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Research

Phosphorylation of B14.5a Subunit from Bovine Heart Complex I Identified by Titanium Dioxide Selective Enrichment and Shotgun Proteomics^*,S

Gabriella Pocsfalvi^,, Manuela Cuccurullo, Gitta Schlosser^,¶, Salvatore Scacco^||, Sergio Papa^|| and Antonio Malorni

From the Proteomic and Biomolecular Mass Spectrometry Centre, Institute of Food Science and Technology, Consiglio Nazionale delle Ricerche, 83100 Avellino, Italy, ^|| Department of Medical Biochemistry, Biology and Physics, University of Bari, 70124 Bari, Italy, and ^¶ Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös L. University, 1518 Budapest, Hungary

Shotgun proteomics was used to study the steady phosphorylation state of NADH:ubiquinone oxidoreductase (complex I) 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 nanoscale reverse-phase HPLC-ESI-MS/MS in single information-dependent acquisition. 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, B., Aggeler, R., Schulenberg, B., Murray, J., Row, R. H., Capaldi, R. A., and Gibson, B. W. (2005) Mass spectrometric identification of novel phosphorylation site in subunit NDUFA10 of bovine mitochondrial complex I. FEBS Lett. 579, 2485–2490). In our work, this finding was 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.

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