Proteomic analysis of the subunit composition of complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria.
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.
Fearnley
Ian M
IM
Dunn Human Nutrition Unit, The Medical Research Council, Cambridge, UK.
Carroll
Joe
J
Walker
John E
JE
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D042967:Electron Transport Complex I / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008929:Mitochondria, Heart / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
D024101:Mitochondrial Proteins; D020543:Proteome; D014357:Trypsin; D042967:Electron Transport Complex I; D003545:Cysteine