Comparison of mitochondrial protein expression in wildtype and mitochondrial branched chain aminotransferase (BCATm) KO mice using isotope coded affinity tags (iTRAQ)

BCATm KOs have increased energy expenditure, higher food intake normalized to body weight, reduced adiposity, resistance to DIO, as well as increased rates of oxygen consumption (VO 2 ) during the light and dark cycle (~32%). Factors frequently associated with altered VO 2 in mice, were not affected in the KO. These included: plasma T4; tissue expression (RT‐PCR) of iodothyronine deiodinase‐2 (D2), UCP‐1, PGC‐1α, β‐adrenergic receptor 3, SERCA1 and glycerol phosphate dehydrogenase; locomotive activity. To verify these and other potential contributors of mitochondrial dysfunction in the KO, the relative expression of proteins in mitochondria isolated from muscle, liver and kidney of KO and wildtype mice was examined. Isolated mitochondrial were digested with trypsin, and the tryptic peptides labeled with one of four iTRAQs (a set of isobaric, multiplex‐able isotope coded affinity tags that can be quantified during mass spectrometry in TOF‐TOF mode). Samples were combined, decomplexed by 2D‐LC and analyzed by MALDI TOF‐TOF. Thousands of iTRAQ‐labeled proteins were identified allowing us to assess the relative concentration of transporters, ATP‐synthase subunits, electron transport chain components, Krebs cycle enzymes, creatine kinase, myokinase, enzymes of lipid and amino acid metabolism, signaling and other proteins that co‐purified with mitochondria in the KO. DK053843 DK062880 DK34738 PA‐DOH