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bioRxiv · 10.1101/2023.06.13.544682

Development of an in situ cell-type specific proteome analysis method using antibody-mediated biotinylation

Ryu, T.·Kim, S.-Y.·Thuraisamy, T.·Jang, Y.·Na, C. H.
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Abstract

Since proteins are essential molecules exerting cellular functions, decoding proteome changes is the key to understanding the normal physiology and pathogenesis mechanism of various diseases. However, conventional proteomic studies are often conducted on tissue lumps, in which multiple cell types are entangled, presenting challenges in interpreting the biological dynamics among diverse cell types. While recent cell-specific proteome analysis techniques, like BONCAT, TurboID, and APEX, have emerged, their necessity for genetic modifications limits their usage. The alternative, laser capture microdissection (LCM), although it does not require genetic alterations, is labor-intensive, time-consuming, and requires specialized expertise, making it less suitable for large-scale studies. In this study, we develop the method for in situ cell-type specific proteome analysis using antibody-mediated biotinylation (iCAB), in which we combined immunohistochemistry (IHC) with the biotin-tyramide signal amplification approach. Poly-horseradish peroxidase (HRP) conjugated to the secondary antibody will be localized at a target cell type via a primary antibody specific to the target cell type and biotin-tyramide activated by HRP will biotinylate the nearby proteins. Therefore, the iCAB method can be applied to any tissues that can be used for IHC. As a proof-of-concept, we employed iCAB for mouse brain tissue enriching proteins for neuronal cell bodies, astrocytes, and microglia, followed by identifying the enriched proteins using 16-plex TMT-based proteomics. In total, we identified [~]8,400 and [~]6,200 proteins from enriched and non-enriched samples. Most proteins from the enriched samples showed differential expressions when we compared different cell type data, while there were no differentially expressed proteins from non-enriched samples. The cell type enrichment analysis with the increased proteins in respective cell types using Azimuth showed that neuronal cell bodies, astrocytes, and microglia data exhibited Glutamatergic Neuron, Astrocyte and Microglia/Perivascular Macrophage as the representative cell types, respectively. The proteome data of the enriched proteins showed similar subcellular distribution as non-enriched proteins, indicating that the iCAB-proteome is not biased toward any subcellular compartment. To our best knowledge, this study represents the first implementation of a cell-type-specific proteome analysis method using an antibody-mediated biotinylation approach. This development paves the way for the routine and widespread use of cell-type-specific proteome analysis. Ultimately, this could accelerate our understanding of biological and pathological phenomena.

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Ryu, T., Kim, S.-Y., Thuraisamy, T., Jang, Y., Na, C. H.. 2023-06-14. Development of an in situ cell-type specific proteome analysis method using antibody-mediated biotinylation. https://doi.org/10.1101/2023.06.13.544682

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