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Contributo in rivista
Tipo: Articolo in rivista
Titolo: Shotgun proteomics for the characterisation of subunit composition of mitochondrial complex I.
Anno di pubblicazione: 2006
Formato: Elettronico Cartaceo
Autori: Pocsfalvi Gabriella; Cuccurullo Manuela; Schlosser Gitta; Cacace Giuseppina; Siciliano Rosa Anna; Mazzeo Maria Fiorella; Scacco Salvatore; Cocco Tiziana; Gnoni Antonio; Malorni Antonio; Papa Sergio
Affiliazioni autori: Proteomic and Biomolecular Mass Spectrometry Centre, Institute of Food Science and Technology, CNR, Avellino Hungarian Academy of Sciences, Eötvös L. University, Budapest Institute of Medical Biochemistry and Chemistry, University of Bari, Bari Institute of Biomembranes and Bioenergetics, Italian National Research Council, Bari
Abstract: Here we propose shotgun proteomics as an alternative method to gel-based bottom-up proteomic platform for the structural characterization of mitochondrial NADH:ubiquinone oxidoreductase (complex I). The approach is based on simultaneous identification of subunits after global digestion of the intact complex. Resulting mixture of tryptic peptides is purified, concentrated, separated and online analyzed using nano-scale reverse-phase nano-ESI-MS/MS in a single information dependent acquisition mode. The usefulness of the method is demonstrated in our work on the well described model system of complex I from bovine heart mitochondria. The shotgun method led to the identification and partial sequence characterization of 42 subunits representing more than 95% coverage of the complex. In particular, almost all nuclear (except MLRQ) and 5 mitochondria DNA encoded subunits (except ND4L and ND6) were identified. Furthermore, it was possible to identify 30 co-purified proteins of the inner mitochondrial membrane structurally not belonging to complex I. The method's efficiency is shown by comparing it to two classical 1 D gel-based strategies. Shotgun proteomics is less laborious, significantly faster and requires less sample material with minimal treatment, facilitating the screening for post-translational modifications and quantitative and qualitative differences of complex I subunits in genetic disorders.
Pagine da: 1438
Pagine a: 1450
Biochimica et biophysica acta. Bioenergetics
Numero volume: 1757
Referee: Sě: Internazionale
Indicizzato da: ISI Web of Science (WOS) 
Altre informazioni: 6
Allegati: Shotgun proteomics for the characterisation of subunit composition of mitochondrial complex I.
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