Comparison of microglial proteomes of MACS and FACS-based purified CD11b+ microglia

ABSTRACT: Background Proteomic characterization of microglia has been limited by low yield and contamination by non-microglial proteins by magnetic-activated cell sorting (MACS) enrichment strategies. To determine whether a fluorescent-activated cell sorting (FACS)-based strategy overcomes these limitations, we compared microglial proteomes of MACS and FACS-based purified CD11b+ microglia in order to identify core sets of microglial proteins in adult mouse brain tissue. Results Quantitative multiplex proteomics by tandem mass tag mass spectrometry (TMT-MS) of MACS-enriched (N = 5) and FACS-purified (N = 5) adult wild-type CD11b+ microglia identified 1,791 proteins, of which 953 were differentially expressed indicating significant differences between both approaches. While the FACS-purified microglia proteome was enriched with cytosolic, endoplasmic reticulum and ribosomal proteins involved in protein metabolism and immune system functions, the MACS-enriched microglia proteome was enriched with proteins related to mitochondrial function and synaptic transmission. As compared to MACS, the FACS microglial proteome showed strong enrichment for canonical microglial proteins while neuron, astrocyte, and oligodendrocyte proteins were decreased. Interestingly, we observed enrichment of endothelial specific proteins in the FACS microglia proteome. By comparing FACS-purified microglia proteomes with transcriptomes, we observed highly concordant as well as highly discordant proteins that were abundant at the protein level but low at the transcript level. Conclusions We demonstrate that TMT-MS proteomics of FACS-purified adult microglia is superior to column-based enrichment approaches, resulting in purer and highly-enriched microglial proteomes. We also define core sets of highly-abundant adult microglial proteins including Moesin (Msn) that can guide future studies.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Microglial Cell

SUBMITTER: Eric Dammer  

LAB HEAD: Nicholas T. Seyfried

PROVIDER: PXD015652 | Pride | 2020-05-11

REPOSITORIES: Pride