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Mitochondria provide energy for biological cells to function, but this process is also a source of oxygen radicals that are capable of damaging nearby proteins. Mitochondrial protein damage can eventually lead to cell death, especially in the case of heart cells, where mitochondria are present in the highest concentrations. As a result, this process is believed to be a cause of many cases of heart disease. In this project, methods for the sensitive detection, isolation and characterization of oxidatively damaged proteins were developed. In the first approach, blue-native polyacrylamide gel electrophoresis (BN-PAGE) was used in combination with liquid chromatography-electrospray-tandem mass spectrometry for efficient isolation and detection of the electron transport chain complexes from mouse heart mitochondria. For the detection of oxidatively damaged proteins, BN-PAGE in conjunction with a hydrazide isotope coded affinity tag (HICAT) was tested. Unfortunately, so far, our findings regarding the use of BN-PAGE in combination with the HICAT probe remain inconclusive. As a second approach for the isolation of oxidatively modified peptides, an alternative strategy was developed which is based on avidin affinity chromatography for the enrichment of biotinylated peptides. This approach was validated with thioredoxin (TRX) as a model protein and led to the identification of the TRX His-6 residue as the primary in vitro adduction site of the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE). |
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