| As both producers and targets of reactive oxygen species, mitochondria undergo oxidative stress as a result of aging. The central hypothesis of this thesis is that mitochondria with different amounts of superoxide production cause different degrees of protein carbonylation with its severity related to proteins' proximity to superoxide production sites. This thesis describes the development of two methods for measuring superoxide abundance and one proteomic method for the identification of carbonylated proteins in mitochondria, which are needed to test the hypothesis.; The first method is based on the micellar electrokinetic chromatography separation and laser induced fluorescence detection of 2-hydroxyethidium, an oxidation product of hydroethidine by superoxide. This method reported that the abundance of 2-hydroxyethidium is 5-times higher outside the matrix of mitochondria isolated from skeletal muscle than inside.; The second method detected the fluorescence of hydroethidine oxidation products in the matrix of individual mitochondria using capillary electrophoresis with laser induced fluorescence detection. Comparing the fluorescence distributions of individual mitochondria isolated from 143B and DeltaH2-1 cells, this method indicated that the residual superoxide levels of mitochondria are higher in the latter than the former.; The proteomic method was based on labeling carbonylated proteins with biotin hydrazide, purifying them by avidin affinity chromatography, digesting them with trypsin, labeling the digested peptides with isobaric stable isotope tags, and analyzing them in a linear ion trap tandem mass spectrometer. Upon database searching and quantitative mass spectrometric analysis, it identified 243 carbonylated proteins from crudely enriched muscle mitochondria.; Finally, this thesis describes a preliminary study that uses these methods to investigate superoxide-induced protein carbonylation in mitochondria. Menadione, inducing superoxide production in vivo, was used to treat isolated mitochondria, which were subsequently analyzed for superoxide production and protein carbonylation. This study reports that NADH dehydrogenase flavoprotein 1 has increased its carbonyl content after the treatment. Since this protein catalyzes the transformation of menadione to semiquinone that generates superoxide, it appears that superoxide causes carbonyl formation to proteins close to superoxide generation sites, one of the two key points of the central hypothesis. In the future, the methods will be critical for further testing this hypothesis. |
