Proteomic analysis of alcohol and LPS-treated primary mouse microglia

Microglia, the resident immune cells of the brain, can exhibit a broad range of activation phenotypes, many of which have been implicated in several diseases and disorders of the central nervous system including alcohol use disorders and disorders. By utilizing a method optimized for sensitive and rapid quantitative proteomic analysis of microglia involving suspension trapping (S-Trap), we were able to produce efficient and reproducible protein extraction from low cell yielding primary mouse brains. Using a ~2 h gradient on a 75 cm UPLC column with a modified data dependent acquisition method on a hybrid quadrupole-Orbitrap mass spectrometer (QE Plus), 5,062 total proteins were identified where 4,928 of those proteins were quantifiable by label-free quantitation (with 5 biological replicates). This analysis resulted in the most comprehensive proteomic dataset for ethanol- and LPS-treated primary mouse microglia to date and even expanded upon the well-characterized macrophage/microglia response to LPS treatment. This study also highlights the subtle, yet significant changes ethanol exposure can induce when compared to control. Interestingly, these changes are not consistent with the robust classical activation induced by LPS treatment, but instead align with the emerging theory that ethanol-treated microglia yield an alternative activation response. The contrast to LPS-treated microglia leads us to conclude that ethanol does not elicit a strong inflammatory response but rather might have a general inhibitory effect on multiple pathways such as phagocytosis and cell migration.