Proteomic analysis of succinate dehydrogenase and ubiquinol-cytochrome c reductase (Complex II and III) isolated by immunoprecipitation from bovine and mouse heart mitochondria.
The oxidative phosphorylation system (OXPHOS) consists of five multi-enzyme complexes, Complexes I-V, and is a key component of mitochondrial function relating to energy production, oxidative stress, cell signaling and apoptosis. Defects or a reduction in activity in various components that make up the OXPHOS enzymes can cause serious diseases, including neurodegenerative disease and various metabolic disorders. Our goal is to develop techniques that are capable of rapid and in-depth analysis of all five OXPHOS complexes. Here, we describe a mild, micro-scale immunoisolation and mass spectrometric/proteomic method for the characterization of Complex II (succinate dehydrogenase) and Complex III (ubiquinol-cytochrome c reductase) from bovine and rodent heart mitochondria. Extensive protein sequence coverage was obtained after immunocapture, 1D SDS PAGE separation and mass spectrometric analysis for a majority of the 4 and 11 subunits, respectively, that make up Complexes II and III. The identification of several posttranslational modifications, including the covalent FAD modification of flavoprotein subunit 1 from Complex II, was possible due to high mass spectrometric sequence coverage.
Schilling
Birgit
B
Buck Institute for Age Research, Novato, CA 94945, USA.
Murray
James
J
Yoo
Chris B
CB
Row
Richard H
RH
Cusack
Michael P
MP
Capaldi
Roderick A
RA
Gibson
Bradford W
BW
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D014450:Electron Transport Complex III / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D051379:Mice; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics; D013385:Succinate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D021122:Protein Subunits; D013385:Succinate Dehydrogenase; D014450:Electron Transport Complex III