Proteome analysis of murine organs using Auto2D

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is a widely used proteomic tool that separates proteins based on the isoelectric points and molecular weights, allowing the observation of hundreds to thousands of proteins in a gel image. Because the protocol for the protein extraction and 2D-PAGE vary between laboratories, data comparison and integration across laboratories are challenging. By standardizing the protocol, collaborative research using the 2D-PAGE can be conducted across multiple laboratories. This study aimed to establish the standardized protocol for the protein extraction and 2D-PAGE. Using an automated 2D-PAGE machine, Auto2D, 620 common spots were identified across protein samples extracted from murine eight organs. Hierarchical cluster analysis based on the intensity of the spots revealed that samples formed groups according to the respective organs. Furthermore, correlation analysis yielded correlation coefficient of ≥0.75 for samples from the same organ. Principal component analysis (PCA) clearly separated samples into distinct clusters, which corresponded to different types of organs. Cluster analysis, correlation analysis, and PCA demonstrated that data was reliably reproducible using the same protein extraction protocol, indicating no need to change the protocol for the protein extraction for each organ. With the standardized protocol for the protein extraction and 2D-PAGE developed in this study, collaborative proteome analysis across multiple laboratories is feasible, enabling reliable data comparison and integration between laboratories and samples.