Fig. 1
A map of the lysosomal proteome and interactome in human neurons and mouse brains. A Schematics of lysosomal proximity labeling (Lyso-APEX) in i3Neurons, lysosomal immunopurification (Lyso-IP) in i3Neurons, and lysosomal biotinylation by antibody recognition (Lyso-BAR) in fixed mouse brain tissues. B Fluorescence imaging of Lyso-APEX, Lyso-IP, and Lyso-BAR activities in i3Neurons and fixed mouse brains. Biotinylated proteins, stained with streptavidin (SA-488), colocalize with lysosomal markers in i3Neurons and fixed mouse brain tissues. HA-tagged lysosomes colocalize with lysosomal markers in i3Neurons. Nuclei were stained by Hoechst. Scale bars are 10 μm. C Volcano plots showing significantly enriched proteins from WT Lyso-APEX compared to cytosolic-APEX, Lyso-IP compared to control neurons without HA-LAMP1 expression, and Lyso-BAR compared to control mouse brains without LAMP1 primary antibody staining (N = 4 for each group). Dotted lines denote corrected p-value of 0.05 (y-axis) and ratio of 1.5 (x-axis). The known lysosomal membrane and lumen proteins are highlighted in blue and orange colors, respectively. D GO enrichment analyses of significantly enriched proteins in Lyso-APEX, Lyso-IP, and Lyso-BAR proteomics. E Venn diagram comparison of significantly enriched proteins in three proteomics methods. F Radar plot comparison of three methods regarding the proteome coverage (lysosome lumen, membrane, and interaction), tag expression level, sensitivity, specificity, and robustness