MassIVE MSV000088822

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Deep single-cell type proteome profiling of mouse brain by nonsurgical AAV-mediated proximity labeling

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Description

Proteome profiling is a powerful tool in biological and biomedical studies, starting with samples at bulk, single cell, or single cell type levels. Reliable methods for extracting specific cell type proteome are in need, especially for the cells (e.g., neurons) that cannot be readily isolated. Here, we present an innovative proximity labeling (PL) strategy for single cell type proteomics of mouse brain, in which TurboID (an engineered biotin ligase) is used to label almost all proteins in a specific cell type. This strategy bypasses the requirement of cell isolation and includes five major steps: (i) constructing recombinant adeno-associated viruses (AAV) to express TurboID driven by cell type-specific promoters, (ii) delivering the AAV to mouse brains by direct intravenous injection, (iii) enhancing PL labeling by biotin administration, (iv) purifying biotinylated proteins followed by on-bead protein digestion, and (v) quantitative tandem-mass-tag (TMT). We first confirmed that TurboID can label a wide range of cellular proteins in human HEK293 cells and optimized the single cell type proteomics pipeline. To analyze specific brain cell types, we generated recombinant AAVs to express TurboID-mCherry fusion proteins, driven by neuron- or astrocyte-specific promoters, and validated the expected cell expression by co-immunostaining of mCherry and cellular markers. Subsequent biotin purification and TMT analysis identified ~10,000 unique proteins from a few micrograms of protein samples with excellent reproducibility. Comparative and statistical analysis indicated these PL proteomes contain cell type specific cellular pathways. Although proximity labeling was originally developed for studying protein-protein interactions and subcellular proteomes, we extend it to efficiently tag the entire proteomes of specific cell types in the mouse brain using TurboID biotin ligase. This simple, effective in vivo approach should be broadly applicable to single cell type proteomics. [doi:10.25345/C5VG50] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: single cell type proteomics ; proximity labeling ; AAV, TMT ; mass spectrometry ; proteomics ; proteome ; brain tissue ; neuron ; astrocyte

Contact

Principal Investigators:
(in alphabetical order) 		Junmin Peng, St. Jude Children's Research Hospital, USA
Submitting User: xilouyi
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