Improved Methodology for Sensitive and Rapid Quantitative Proteomic Analysis of Adult‐Derived Mouse Microglia: Application to a Novel In Vitro Mouse Microglial Cell Model

Abstract

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.