title: Shotgun proteomics for the characterization of subunit composition of mitochondrial complex I.
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.
authors.affiliation: Proteomic and Biomolecular Mass Spectrometry Centre, Institute of Food Science and Technology, CNR, via Roma 52, a/c, 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: Giuseppina
authors.initials: G
authors.lastname: Cacace
authors.forename: Rosa Anna
authors.initials: RA
authors.lastname: Siciliano
authors.forename: Maria Fiorella
authors.initials: MF
authors.lastname: Mazzeo
authors.forename: Salvatore
authors.initials: S
authors.lastname: Scacco
authors.forename: Tiziana
authors.initials: T
authors.lastname: Cocco
authors.forename: Antonio
authors.initials: A
authors.lastname: Gnoni
authors.forename: Antonio
authors.initials: A
authors.lastname: Malorni
authors.forename: Sergio
authors.initials: S
authors.lastname: Papa
mesh_terms: D000818:Animals; D002417:Cattle; D042967:Electron Transport Complex I / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008929:Mitochondria, Heart / Q000737:chemistry*; D010449:Peptide Mapping; D021122:Protein Subunits / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D021122:Protein Subunits; D042967:Electron Transport Complex I