Comparative proteomics of flotillin-rich Triton X-100-insoluble lipid raft fractions of mitochondria and synaptosome from mouse brain

There is increasing evidence that lipid rafts may play important roles in brain neuronal cell functions including signal transduction. Meanwhile, the results suggesting possible presence of rafts in intracellular organelles such as mitochondria have been also reported. In this study, we compared proteins in raft-like structure of mitochondria to those of synaptosomal rafts and analyzed age-related alterations in protein in both mitochondrial and synaptosomal lipid rafts. A low density Triton X-100-insoluble fraction was prepared from cerebral cortical synaptosome and mitochondria of mouse and analyzed by two-dimensional (2-D) gel electrophoresis in combination with mass spectrometry. Co-localization of flotillin and cholesterol in Triton X-100-insoluble fraction of mitochondria was shown by Western blot analysis. Differential display of proteins using computer-aided image analysis revealed that the composition of protein in flotillin-rich Triton X-100-insoluble fraction of mitochondria was similar to that found in synaptosome. Several protein spots on 2-D gels varied in quantity depending on the age of the mouse, including the guanine nucleotide-binding protein G(O) alpha subunit, as identified by peptide mass fingerprinting.