Deep Single-Cell-Type Proteome Profiling of Mouse Brain by Nonsurgical AAV-Mediated Proximity Labeling

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 proteomes
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 (AAVs) 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) labeling. We first confirmed that TurboID can label a wide range
of cellular proteins in human HEK293 cells and optimized the single-cell-type
proteomic pipeline. To analyze specific brain cell types, we generated
recombinant AAVs to coexpress TurboID and mCherry proteins, driven
by neuron- or astrocyte-specific promoters and validated the expected
cell expression by coimmunostaining 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 analyses indicated that
these PL proteomes contain cell-type-specific cellular pathways. Although
PL was originally developed for studying protein–protein interactions
and subcellular proteomes, we extended 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.