Fig. 1

Study design and analytic approach for comprehensive quantitative proteomic analysis of isolated mouse microglia. a Workflow summarizing isolation and purification of CD11b⁺ brain immune cells from 4-month-old male C57Bl6/J wild-type mice (N = 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, N = 5 mice) or isolated via fluorescent activated cell sorting (FACS, N = 5 mice). Representative flow cytometry gating strategy and antibody separation using CD11b-APC/Cy7 antibody for isolation of microglia. b 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 (n = 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