Figure 3. Isolation-independent methods for in vivo proteomic labeling of microglia.

In vivo biorthogonal amino acid tagging (BONCAT) of proteins (red panel) and cell type-specific in vivo biotinylation of proteins (CIBOP) (blue panel) is achieved by inserting mutant MetRS (L274G) or MetRS*, or TurboID, into the Rosa26 locus to generate in vivo proteomic labeling in mouse models. Following, microglia specificity can be achieved by either breeding the MetRS* or TurboID models with a microglia-specific Cre mouse, with or without inducibility, or by injecting a microglial-specific adeno-associated virus (AAV) to deliver Cre. MetRS* contains a mutation (L247G) in the amino acid binding site which tags nascent proteins with an azide tagged methionine analog, azidonorleucine (ANL). The azide residue of ANL can undergo “click” chemistry in which ANL-tagged proteins residues are “clicked” with a PEG-biotin-alkyne. Proximity labeling using CIBOP is achieved by the biotin ligase, TurboID, that biotinylates endogenous proteins in close proximity. After, MetRS* or TurboID tagged proteins can undergo biotin affinity capture using streptavidin-coated beads and processed for downstream mass MS-based proteomics.