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

Gabriella Pocsfalvis, Manuela Cuccurullo, G. Schlosser, Salvatore Scacco, Sergio Papa, Antonio Malorni

Research output: Contribution to journalArticle

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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 1314.5a. 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 NDUFA1O of bovine mitochondrial complex I. FEBS Left. 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.

Original languageEnglish
Pages (from-to)231-237
Number of pages7
JournalMolecular and Cellular Proteomics
Volume6
Issue number2
DOIs
Publication statusPublished - Feb 2007

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Phosphorylation
Firearms
Proteomics
Phosphopeptides
Electron Transport Complex I
Heart Mitochondria
Mitochondria
Phosphoproteins
Mass spectrometry
Digestion
Mass Spectrometry
Gels
High Pressure Liquid Chromatography
titanium dioxide
Ions
Staining and Labeling
Peptides
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Phosphorylation of B14.5a subunit from bovine heart complex I identified by titanium dioxide selective enrichment and shotgun proteomics. / Pocsfalvis, Gabriella; Cuccurullo, Manuela; Schlosser, G.; Scacco, Salvatore; Papa, Sergio; Malorni, Antonio.

In: Molecular and Cellular Proteomics, Vol. 6, No. 2, 02.2007, p. 231-237.

Research output: Contribution to journalArticle

Pocsfalvis, Gabriella ; Cuccurullo, Manuela ; Schlosser, G. ; Scacco, Salvatore ; Papa, Sergio ; Malorni, Antonio. / Phosphorylation of B14.5a subunit from bovine heart complex I identified by titanium dioxide selective enrichment and shotgun proteomics. In: Molecular and Cellular Proteomics. 2007 ; Vol. 6, No. 2. pp. 231-237.
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