全自動2次元電気泳動装置を用いたグリオーマ特異的蛋白質群の解析

抄録

We have developed a fully automated two-dimensional gel electrophoresis system (auto-2D system) which makes tissue/cellular protein analysis by 2D-PAGE and 2D-western blotting more simple, quick, sensitive, and reproducible with a small volume of sample within 2 hrs, thus, being expected as a convenient clinical analysis tool for the personal diagnosis. In this study, we utilized this system for the analysis of brain tumor specific proteins. Malignant gliomas are generally resistant to all standard therapies, however, patients with Anaplastic oligodendroglioma(AOG) or oligoastrocytoma(AOA) with loss of heterozygosity on chromosome 1p/19q (LOH+) frequently respond to chemotherapies. Information of the factors for these clinical findings has not been reported so fat. We have been studying specific proteins related to these findings by using newly established techniques of proteomics such as phospho-2D-DIGE and iTRAQ methods. Recently, we identified 105 specific proteins expressing in LOH- compared with LOH+AOG/AOA, which include EGFR-MAPK/PI3K-AKT signal molecules, vascular PIFs, adhesion molecules, cell cycle regulator CDK families, and cytoskeletal regulating proteins. Among them, we focused on vimentin, GFAP and CDK4 that were highly expressed with unique 17 phosphorylated/proteolysed forms in LOH- samples. Using a specific antibody cocktail to detect these proteins by the auto-2D-western blotting, we analyzed 30 samples of AOG/AOA and identified the specific pattern of LOH- tissue compared with LOH+ samples. The resolution, sensitivity and reproducibility for the analysis of this brain tissue specific spot pattern by the auto-2D method were significantly higher than those by the conventional methods. These results suggest that this auto-2D-western method will be useful to study the specific protein expression profiles not only for the brain tumors but also for variety of the biological specimens, and could be introduced as a new clinical laboratory technology.