title: Phosphorylation of B14.5a subunit from bovine heart complex I identified by titanium dioxide selective enrichment and shotgun proteomics.
abstract: 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.
authors.affiliation: Proteomic and Biomolecular Mass Spectrometry Centre, Institute of Food Science and Technology, Consiglio Nazionale delle Ricerche, 83100 Avellino, Italy. gpocsfalvi@isa.cnr.it
authors.forename: Gabriella
authors.initials: G
authors.lastname: Pocsfalvi
authors.forename: Manuela
authors.initials: M
authors.lastname: Cuccurullo
authors.forename: Gitta
authors.initials: G
authors.lastname: Schlosser
authors.forename: Salvatore
authors.initials: S
authors.lastname: Scacco
authors.forename: Sergio
authors.initials: S
authors.lastname: Papa
authors.forename: Antonio
authors.initials: A
authors.lastname: Malorni
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D042967:Electron Transport Complex I / Q000737:chemistry / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D010766:Phosphorylation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014025:Titanium / Q000494:pharmacology*; D014451:Ubiquinone / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D021122:Protein Subunits; D009243:NAD; D014451:Ubiquinone; C009495:titanium dioxide; D014025:Titanium; D010088:Oxidoreductases; D042967:Electron Transport Complex I