Characterization of Mouse Spleen Cells by Subtractive Proteomics*S

Major analytical challenges encountered by shotgun proteome analysis include both the diversity and dynamic range of protein expression. Often new instrumentation can provide breakthroughs in areas where other analytical improvements have not been successful. In the current study, we utilized new instrumentation (LTQ FT) to characterize complex protein samples by shotgun proteomics. Proteomic analyses were performed on murine spleen tissue separated by magnetic beads into distinct CD45− and CD45+ cell populations. Using shotgun protein analysis we identified ∼2,000 proteins per cell group by over 12,000 peptides with mass deviations of less than 4.5 ppm. Datasets obtained by LTQ FT analysis provided a significant increase in the number of proteins identified and greater confidence in those identifications and improved reproducibility in replicate analyses. Because CD45− and not CD45+ cells are able to regenerate functional pancreatic islet cells in a mouse model of type I diabetes, protein expression was further compared by a subtractive proteomic approach in search of an exclusive protein expression profile in CD45− cells. Characterization of the proteins exclusively identified in CD45− cells was performed using gene ontology terms via the Javascript GoMiner. The CD45− cell subset readily revealed proteins involved in development, suggesting the persistence of a fetal stem cell in an adult animal.