Towards complete proteome coverage of microglia in a 2-hour, single-shot analysis: characterization of microglial response to acute alcohol exposure
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Issue Date
2020-07
Authors
Wohlfahrt, Jessica '20
Degree
Advisor
Stevens, Stanley
Committee Members
Balaz, Stephan
Voigt, Jeffrey
Voigt, Jeffrey
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Abstract
Alcohol abuse results in harmful effects on the central nervous system, including a reported induction of a pro-inflammatory phenotype in microglia; however, emerging evidence suggests a complex range of not fully characterized microglial activation phenotypes involving differential activation of phagocytosis. Due to the complex nature of alcohol-induced microglial activation, the purpose of this study was to develop and utilize an improved mass spectrometry-based proteomics to produce more comprehensive proteome coverage in a single-shot analysis.
Methodology developed using BoxCar data acquisition (Mann lab) was performed on adult-derived mouse microglia (IMG cells) and primary neonatal-derived mouse microglia. High pH reversed-phase offline fractionation followed by mass spectrometric analysis of pooled control and pro-inflammatory IMG (LPS-treated) lysate resulted in the identification of 8,537 unique proteins. The same analysis on control, LPS-, and ethanol-treated pooled primary microglia lead to the identification of 7,959 unique proteins. Control and LPS-treated IMG cells as well as control, LPS-, and ethanol-treated primary cells with TREM2 (a phagocytosis regulator) agonist cotreatment were analyzed in triplicate using BoxCar data acquisition and the results were searched using MaxQuant with the match-between-runs feature enabled for comparison to the original fractionation dataset. The BoxCar approach resulted in the identification of 7,074 ± 46 IMG proteins and 4323 ± 239 MM proteins in a 2-hr single-shot analysis, which is an approximate 40% improvement in identification over conventional data acquisition.
To the best of our knowledge, this study highlights the deepest proteome coverage to date for microglia and represents a significant methodological advancement in terms of rapid and comprehensive characterization of the neuroimmune response in alcohol exposure models. This study was also able to identify proteins such as SIRT2 and DAB2 which may be involved in TREM2 receptor activation. These TREM2 agonist effects seem to be blunted by ethanol.
Citation
Wohlfahrt J. Towards complete proteome coverage of microglia in a 2-hour, single-shot analysis: characterization of microglial response to acute alcohol exposure. Ann Arbor (MI): Proquest LLC; 2020. 59 p.
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