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Background Microglia are the resident immune cells in the brain where they play essential roles in the development and maintenance of physiological functions of this organ. Aberrant activation of microglia is speculated to be involved in the pathogenesis of a variety of neurological disorders, including alcohol use disorders. Repeated binge ethanol (EtOH) consumption can have a profound impact on the function and integrity of the brain resulting in changes in behaviors such as withdrawal and reward. However, the microglial molecular and cellular pathways associated with EtOH binge consumption remain poorly understood. Method In this study, adult C57BL/6J male and female mice were subjected daily to a gelatin‐based drinking‐in‐the‐dark voluntary EtOH consumption paradigm (3 h/d for 4 months) to characterize EtOH consumption and withdrawal‐associated and anxiety‐like behaviors. Brain microglia were isolated at the end and analyzed for protein expression profile changes using unbiased mass spectrometry‐based proteomic analysis. Results Both male and female mice consistently consumed binge quantities of EtOH daily, resulting in blood EtOH levels > 80 mg/dl measured at the end of the 3‐hour daily consumption period. Although female mice consumed a significantly greater amount of EtOH than male mice, EtOH withdrawal‐associated anxiety‐like behaviors measured by marble‐burying, light‐dark box, and elevated plus maze tests were predominantly observed in male mice. Proteomic analysis of microglia isolated from the brains of animals at the end of the 4‐month binge EtOH consumption identified 117 and 37 proteins that were significantly up‐ or downregulated in EtOH‐exposed male and female mice, respectively, compared to their pair‐fed controls. Protein expression profile‐based pathway analysis identified several cellular pathways that may underlie the sex‐specific and EtOH withdrawal‐associated behavioral abnormalities. Conclusion Taken together, our findings revealed sex‐specific changes in EtOH withdrawal‐associated behaviors and signaling pathways in the mouse brain microglia and may help advance our understanding of the molecular, cellular, and behavioral changes related to human binge EtOH consumption.

Microglia, as the resident brain immune cells, can exhibit a broad range of activation phenotypes, which have been implicated in a multitude of central nervous system disorders. Current widely studied microglial cell lines are mainly derived from neonatal rodent brain that can limit their relevance to homeostatic function and disease-related neuroimmune responses in the adult brain. Recently, an adult mouse brain-derived microglial cell line has been established; however, a comprehensive proteome dataset remains lacking. Here, an optimization method for sensitive and rapid quantitative proteomic analysis of microglia is described that involves suspension trapping (S-Trap) for efficient and reproducible protein extraction from a limited number of microglial cells expected from an adult mouse brain (300 000). 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, 4855 total proteins have been identified where 4698 of which are quantifiable by label-free quantitation with a median and average coefficient of variation (CV) of 6.7% and 10.6%, respectively. This dataset highlights the high depth of proteome coverage and related quantitation precision of the adult-derived microglial proteome including proteins associated with several key pathways related to immune response. Data are available via ProteomeXchange with identifier PXD012006.Microglia play a critical role in mediating homeostatic and neuroimmune responses within the central nervous system (CNS). [1] Given the wide spectrum of microglial activation phenotypes that can be exhibited in response to various stimuli, characterizing the

Trapped ion-mobility spectrometry (TIMS) was used to fractionate ions in the gas phase based on their ion mobility (V s/cm2), followed by parallel accumulation–serial fragmentation (PASEF) using a quadrupole time-of-flight instrument to determine the effect on the depth of proteome coverage. TIMS fractionation (up to four gas-phase fractions) coupled to data-dependent acquisition (DDA)-PASEF resulted in the detection of ∼7000 proteins and over 70,000 peptides overall from 200 ng of human (HeLa) cell lysate per injection using a commercial 25 cm ultra high performance liquid chromatography (UHPLC) column with a 90 min gradient. This result corresponded to ∼19 and 30% increases in protein and peptide identifications, respectively, when compared to a default, single-range TIMS DDA-PASEF analysis. Quantitation precision was not affected by TIMS fractionation as demonstrated by the average and median coefficient of variation values that were less than 4% upon label-free quantitation of technical replicates. TIMS fractionation was utilized to generate a DDA-based spectral library for downstream data-independent acquisition (DIA) analysis of lower sample input using a shorter LC gradient. The TIMS-fractionated library, consisting of over 7600 proteins and 82,000 peptides, enabled the identification of ∼4000 and 6600 proteins from 10 and 200 ng of human (HeLa) cell lysate input, respectively, with a 20 min gradient, single-shot DIA analysis. Data are available in ProteomeXchange: identifier PXD033129.