Flow-cytometric microglial sorting coupled with quantitative proteomics identifies moesin as a highly-abundant microglial protein with relevance to Alzheimer’s disease

摘要

Study design and analytic approach for comprehensive quantitative proteomic analysis of isolated mouse microglia. Workflow summarizing isolation and purification of CD11b brain immune cells from 4-month-old male C57Bl6/J wild-type mice (  = 10). Following mechanical dissociation of fresh, whole mouse brains and percoll density centrifugation, mononuclear cells were enriched for CD11b microglia cells by magnetic activating cell sorting (MACS,  = 5 mice) or isolated via fluorescent activated cell sorting (FACS,  = 5 mice). Representative flow cytometry gating strategy and antibody separation using CD11b-APC/Cy7 antibody for isolation of microglia. Proteomic workflow for tandem mass tag (TMT) mass spectrometry (MS) based quantification. All 10 microglia samples were lysed in 8 M urea, digested with LysC and Trypsin, and peptides were labeled using one 10-plex TMT kit. A total of 5 individual MACS-enriched microglia samples were dedicated to the first five channels (126, 127 N, 127C, 128 N, 128C) and 5 individual FACS-isolated microglia samples were dedicated to the last five channels (129 N, 129C, 130 N, 130C, 131). After labeling, the samples were combined and fractionated by off-line high pH fractionation (  = 9 fractions [fx]). Each fraction was analyzed and quantified by synchronous precursor selection (SPS)-MS3 on an Orbitrap Fusion mass spectrometer. Peptide search on raw files were conducted with Proteome Discoverer (v2.1) in order to obtain the MACS microglia proteome and FACS microglia proteome