Proteomic analysis of the subunit composition of complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria.

TitleProteomic analysis of the subunit composition of complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria.
Publication TypeJournal Article
Year of Publication2007
AuthorsFearnley, IM, Carroll, J, Walker, JE
JournalMethods Mol Biol
Volume357
Pagination103-25
Date Published2007
ISSN1064-3745
KeywordsAnimals, Cattle, Chromatography, High Pressure Liquid, Cysteine, Electron Transport Complex I, Electrophoresis, Gel, Two-Dimensional, Electrophoresis, Polyacrylamide Gel, Isoelectric Focusing, Mitochondria, Heart, Mitochondrial Proteins, Protein Processing, Post-Translational, Proteome, Proteomics, Reproducibility of Results, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Tandem Mass Spectrometry, Trypsin
Abstract

Complex I from the inner membranes of mammalian mitochondria is a complicated membrane-bound assembly of redox centers (flavin mononucleotide cofactor, iron sulphur centers) and at least 46 different proteins. The hydrophobic nature of its membrane-bound subunits and the complexity of subunit content present a substantial analytical challenge. The complete protein chemical analysis of complex I purified from bovine mitochondria required the resolution of subunits by one-dimensional and two-dimensional electrophoresis, reverse-phase chromatography, and combinations of these techniques. These subunits were characterized by mass spectrometry (MS)-based protein identification methods, requiring both peptide mass fingerprinting and amino acid sequencing by tandem MS. The components were identified also and characterized by measurements of subunit molecular mass. These strategies have provided a comprehensive view of the subunit content of the intact complex, its structural domains, and stable subunit modifications.

DOI10.1385/1-59745-214-9:103
Alternate JournalMethods Mol. Biol.
Citation Key10.1385/1-59745-214-9:103
PubMed ID17172683
Grant ListMC_U105663148 / / Medical Research Council / United Kingdom