Cellular proteomic profiling using proximity labelling by TurboID-NES in microglial and neuronal cell lines.

Different brain cell types play distinct roles in brain development and disease via cellular mechanisms. Molecular characterization of these cellular mechanisms using cell type-specific approaches, particularly at the protein (proteomic) level, can provide biological and therapeutic insights. Conventional approaches to investigate cell type-specific proteomes from brain pose several technical barriers. To overcome these, in vivo proteomic labeling with proximity dependent biotinylation of cytosolic proteins using TurboID with a Nuclear Export Sequence (TurboID-NES), coupled with mass spectrometry (MS) of labeled proteins, has emerged as a powerful strategy to sample cell type-specific proteomes in the native state of cells without need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID-NES approach are representative of the whole cell proteome, and capture cellular responses to stimuli without disruption of cellular processes. We generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing TurboID-NES to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-NES expression and biotinylation did not significantly impact homeostatic cellular proteomes of BV2 and N2A cells, and did not affect cytokine production or mitochondrial respiration of BV2 cells under resting or lipopolysaccharide (LPS)-stimulated conditions. TurboID-NES mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under resting conditions. Acute LPS treatment significantly altered microglial proteomes, but not N2A proteomes, and the LPS effect was partly captured by analysis of the TurboID-NES-labeled proteome of BV2 cells.