Proteonomic techniques applied on skeletal muscle and fibroblasts in the patients with oxphos defiencies.

Finding the underlying molecular defect in the patients with deficient activity in one of the OXPHOS complexes is not an easy task. Complementation studies can be used to decide whether mutation analysis should be performed in mtDNA or in nuDNA. These studies, however, can be laborious. A diagnostic strategy was developed based on the study of the abundance of the complexes and their subunits for prediction of the location of the primary defect. Immunohisto-chemical and immunocytochemical analysis was performed in skeletal muscle and cultured skin fibroblasts using specific antibodies against OXPHOS subunits. In isolated mitochondria, OXPHOS complexes were separated by blue native polyacrylamide gel electrophoresis (BN-PAGE) and estimation of the abundance of each complex was performed using immunoblotting techniques. Two-dimensional electrophoresis (BN-PAGE/SDS-PAGE) was applied for separation of the subunits. Spots on the 2D gels were identified by immunostaining and mass spectrometric sequence analysis. mtDNA mutations affect the intramitochondrial protein synthesis. These mutations result in a reduced immunoreactivity in BN-PAGE gels for at least two complexes (I and IV mostly), and in a mosaic pattern on immunostaining of tissues and cells (using antibodies against complex I and IV). Mutations in the nuclearly located assembly genes like SURF-1, BCS1L and ATP12 and in the nuclear genes coding for structural proteins were associated with a reduced immunoreactivity of only one complex, that was generalized. The combination of these proteomic techniques can diminish the number of relevant genetic tests and facilitate the identification of the underlying gene defect.