Two-dimensional electrophoresis and mass spectrometric identification of mitochondrial proteins from an SH-SY5Y...

To probe the mitochondrial involvement in neurodegenerative processes, we have generated a high-resolution map of the mitochondrial proteome from a human neuroblastoma SH-SY5Y cell line that has been used for creating cytoplasmic hybrid cell systems. Two mitochondrial preparations were evaluated using two-dimensional (2D) gel electrophoresis and mass spectrometry; one obtained from differential centrifugation and the other by a multiple-step percoll/metrizamide gradient. The 2D gel maps prepared from these mitochondrial fractions separated over 300 distinct spots as visualized by colloidal Coomassie blue (CCB), or closer to 400 proteins with silver staining. The most abundant proteins identified in the mitochondrial fraction prepared by differential centrifugation were those of mitochondrial, cytoplasmic, and endoplasmic reticulum origin. Proteins obtained using the more intensive two-step gradient method were almost exclusively known to be associated with mitochondria. From this latter preparation, 84 of the most abundant gel spots were analyzed, out of which 61 proteins were identified. The absence of many membrane-associated proteins known to be associated with the mitochondrion and the limited number of total proteins observed in the 2D gel maps suggest that the majority of mitochondrial proteins are not being detected under these separation and staining conditions. An insoluble pellet obtained after solubilization of the mitochondrial fraction prepared with the percoll/metrizamide gradient was boiled in sodium dodecylsulfate (SDS) and separated by 1D sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This separation yielded some additional proteins, many of which are likely membrane-associated. These studies form the basis for the analysis of differential protein expression in cybrid cellular models of neurodegenerative disorders and in affected tissue from diseased states.