Abstract
The central nervous system (CNS) comprises diverse and morphologically complex cells. To understand the molecular basis of their physiology, it is crucial to assess proteins expressed within intact cells. Commonly used methods utilize cell dissociation and sorting to isolate specific cell types such as neurons and astrocytes, the major CNS cells. Proteins purified from isolated cells are identified by mass spectrometry-based proteomics. However, dissociation and cell-sorting methods lead to near total loss of cellular morphology, thereby losing proteins from key relevant subcompartments such as processes, end feet, dendrites and axons. Here we provide a systematic protocol for cell- and subcompartment-specific labeling and identification of proteins found within intact astrocytes and neurons in vivo. This protocol utilizes the proximity-dependent biotinylation system BioID2, selectively expressed in either astrocytes or neurons, to label proximal proteins in a cell-specific manner. BioID2 is targeted genetically to assess the subproteomes of subcellular compartments such as the plasma membrane and sites of cell–cell contacts. We describe in detail the expression methods (variable timing), stereotaxic surgeries for expression (1–2 d and then 3 weeks), in vivo protein labeling (7 d), protein isolation (2–3 d), protein identification methods (2–3 d) and data analysis (1 week). The protocol can be applied to any area of the CNS in mouse models of physiological processes and for disease-related research.
Key points
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Astrocyte- and neuron-specific proximity-dependent biotinylation enables identification of a cell’s subproteome by targeting BioID2 to the cytosol using adeno-associated virus plasmid vectors, or to the plasma membranes using an Lck plasma membrane tag.
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Alternative in vivo proximity-dependent labeling approaches are TurboID, genetically targeted horseradish peroxidase and ascorbate peroxidase. Here, two BioID2 enzymes are fused to increase the catalytic activity of the approach.
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Data availability
The authors declare that the main data and all the raw source data discussed in this protocol are available in the supporting primary research paper6. The raw datasets are available at the Proteomics Identification Database with accession identifier PXD029257. Any data are available for research purposes from the corresponding authors upon reasonable request.
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Acknowledgements
J.S.S. was supported by the National Science Foundation Graduate Research Fellowship Program (NSF-GRFP; DGE-2034835) and by the UCLA Eugene V. Cota-Robles Fellowship. B.S.K., J.S.S. and this work were supported by the National Institutes of Health (R35 NS111583, R01 AG075955, R01 DA047444), by an Allen Distinguished Investigator Award, a Paul G. Allen Frontiers Group advised grant of the Paul G. Allen Family Foundation and by the Ressler Family Foundation (to B.S.K.).
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J.S.S. wrote the first draft of the text and made the figures. Y.J.-A. wrote sections on proteomic data analyses. B.S.K. and J.S.S. finalized the text and figures. All authors contributed to the final version.
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Key reference using this protocol
Soto, J. S. et al. Nature 616, 764–773 (2023): https://doi.org/10.1038/s41586-023-05927-7
Extended data
Extended Data Fig. 1 In vivo protein biotinylation in mouse.
a. Photo showing site of subcutaneous biotin injection in mouse for in vivo BioID2 protein biotinylation. b. A successful injection will produce a temporary pocket of biotin under the skin of the mouse (red arrows. The pocket should disappear within 10 minutes.
Extended Data Fig. 2 Microdissection setup and tissue homogenization.
a. Photos depicting the microdissection area for removal and isolation of the CNS region of interest. Because the sample must remain cold, the dissection is conducted with a pre-frozen TissueTek cold plate. Standard microdissection tools for striatum are shown. b. Photos of striata in a dounce homogenizer tube that was prefilled with 600 µL of lysis buffer 1 (Step 126) prior to homogenization with pestles. Photo on the right shows homogenate after homogenization with pestles (Step 127).
Supplementary Information
Supplementary Code 1
Contrast file for artMS analysis input.
Supplementary Code 2
Keys file for artMS analysis input.
Supplementary Code 3
Yaml configuration file for artMS analysis input.
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Soto, J.S., Jami-Alahmadi, Y., Wohlschlegel, J.A. et al. In vivo identification of astrocyte and neuron subproteomes by proximity-dependent biotinylation. Nat Protoc 19, 896–927 (2024). https://doi.org/10.1038/s41596-023-00923-7
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DOI: https://doi.org/10.1038/s41596-023-00923-7
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