Phosphorylation of B14.5a subunit from bovine heart complex I identified by titanium dioxide selective enrichment and shotgun proteomics

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

Biophenol-Protein Supramolecular Models by Fast Atom Bombardment-Mass Spectrometric Experiments

Biomimetic supramolecular formation between hydroxytyrosol, a biophenol found in olives and virgin olive oil, and caffeine or Asp-Phe, as proteic models, has been achieved by FAB-mass spectrometric experiments. The protonated supermolecules show a consistently higher difference in stability constants, thus indicating a preferential molecular recognition site provided by caffeine, the biomimetic model of proline-rich mucoproteins. The spontaneous aggregation of the complementary supramolecular components suggests correlations with the sensorial response and the bioavailability of food biophenols

Microheterogeneity characterization of a trichorzianine-A mixture from Trichoderma harzianum

The microheterogeneity of trichorzianine-A (TA) from Trichoderma harzianum was studied using single-stage mass spectrometry and high-energy collision-induced dissociation (CID) tandem mass spectrometry. Based on the electrospray ionization and the liquid secondary ion single-state mass spectra, the main components with eight different molecular masses were identified in the mixture. Five contain phenylalaninol and three a tryptophanol C-terminal amino alcohol residue. The tandem mass spectra taken on the protonated molecular ion of these components (MH+) show that most of them contain one main and one or more minor sequence analogues. For the microheterogeneity characterization of this mixture, a previously described method was used based on high-energy CID studies of the different MH+, y(7), b(12) ions and their satellite ions with +/-14 and +/-28 mass number differences. In addition to the nine known sequence variants, five new trichorzianines were sequenced from the mixture. It was found, furthermore, that amino acid exchange can occur not only at positions 5, 14, 16 and 19 but also at positions 2, 3, 4, 7, 8 and 9