Dataset Information

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): Orbitrap Fusion

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

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Dataset's files

Source:

			Action	DRS
	mouseUNIPROT_Aug2015.fasta	Fasta
	sr_microgliatmt_sps_01.raw	Raw
	sr_microgliatmt_sps_02.raw	Raw
	sr_microgliatmt_sps_03.raw	Raw
	sr_microgliatmt_sps_04.raw	Raw

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Publications

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

Rayaprolu Sruti S Gao Tianwen T Xiao Hailian H Ramesha Supriya S Weinstock Laura D LD Shah Jheel J Duong Duc M DM Dammer Eric B EB Webster James A JA Lah James J JJ Wood Levi B LB Betarbet Ranjita R Levey Allan I AI Seyfried Nicholas T NT Rangaraju Srikant S

Molecular neurodegeneration 20200507 1

<h4>Background</h4>Proteomic characterization of microglia provides the most proximate assessment of functionally relevant molecular mechanisms of neuroinflammation. However, microglial proteomics studies have been limited by low cellular yield and contamination by non-microglial proteins using existing enrichment strategies.<h4>Methods</h4>We coupled magnetic-activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) of microglia with tandem mass tag-mass spectrometry (TMT-MS) ...[more]

PMID: 32381088

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