Fig. 1: Development and validation of the Rosa26TurboID/wt mouse line for cell type-specific proteomics.
a Genetic strategy for targeting TurboID (V5-TurboID-NES) to the Rosa26 locus. b Schematic of AAV studies to direct Cre recombinase expression in hippocampal neurons (n = 3 WT mice with no injections, n = 3 WT mice received AAV9-hSyn-Cre, n = 3 Rosa26TurboID/wt mice received AAV9-hSyn-Cre). c Representative immunofluorescence images of CA2/3 of the hippocampus from WT/hSyn (n = 3 mice) and Rosa26TurboID/wt/hSyn (n = 3 mice) showing biotinylation (green: streptavidin Alexa488) in relation to neuronal nuclei (magenta: NeuN). d Western blot of brain lysates (representative animal from n = 3 mice/group) probed with streptavidin fluorophore, anti-V5, and anti-Gapdh antibodies. Rosa26TurboID/wt/hSyn brain showed biotinylated proteins of different molecular weights as compared to few endogenously biotinylated proteins in the two control groups. Right: Densitometry confirming significant increase in biotinylation signal in Rosa26TurboID/wt/hSyn brains (one-way ANOVA, ****p = 0.0001; data represented as mean ± SEM). e Western blot (left) and silver stain (right) of enriched biotinylated proteins after streptavidin-pulldown and release of biotinylated proteins from 10% of streptavidin beads (representative images from n = 3 mice/group). As compared to minimal protein enriched in the two control groups, several biotinylated proteins were enriched from Rosa26TurboID/wt/hSyn brain. f Volcano plots showing differentially enriched proteins comparing Rosa26TurboID/wt/hSyn (n = 3) and control mice (n = 3 WT and n = 3 WT/hSyn). Orange symbols (two-sided T-test unadjusted p ≤ 0.05 and ≥2-fold change) represent biotinylated proteins enriched in the Rosa26TurboID/wt/hSyn brain and examples of neuron-specific proteins are highlighted, in addition to TurboID. Blue symbols represent endogenously biotinylated carboxylases enriched in the control brains. For group wise comparisons, see Supplementary Fig. 1. g Results from GSEA of ≥2-fold biotinylated neuronal proteins (orange symbols from panel f), as compared to reference list5 (mouse brain: n = 7736) showed enrichment of neuronal and synaptic proteins confirming neuron-specific labeling. h Graphical representation of the number of proteins within various cellular compartments determined from GSEA. For related MS data and additional analyses, see Supplementary Data 1, 3, 7, and 8. Source data are provided as a Source Data file.
