Mass spectrometric characterization of mitochondrial electron transport complexes: subunits of the rat heart ubiquinol-cytochrome c reductase.
Complex III of the mitochondrial electron transport chain, ubiquinol-cytochrome c reductase, was isolated by blue native polyacrylamide gel electrophoresis. Ten of the 11 polypeptides present in this complex were detected directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following electroelution of the active complex. Tryptic and chymotryptic digestion of the complex permit the identification of specific peptides from all of the protein subunits with 70% coverage of the 250 kDa complex. The mass of all 11 proteins was confirmed by second dimension Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and elution of the separated polypeptides. Additionally, the identity of the core I, core II, cytochrome c and the Rieske iron-sulfur protein were confirmed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) characterization of the peptides generated by in-gel trypsin digestion of the SDS-PAGE separated proteins. The methodology demonstrated for analyzing this membrane-bound electron transport complex should be applicable to other membrane complexes, particularly the other mitochondrial electron transport complexes. The MS analysis of the peptides obtained by in-gel digestion of the intact complex permits the simultaneous characterization of the native proteins and modifications that contribute to mitochondrial deficits that have been implicated as contributing to pathological conditions.
Sun
Gang
G
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohis 44106, USA.
Kinter
Michael T
MT
Anderson
Vernon E
VE
D000595:Amino Acid Sequence; D000818:Animals; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008929:Mitochondria, Heart / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D021122:Protein Subunits; D014357:Trypsin; D014450:Electron Transport Complex III