Cellular proteomic profiling using proximity labeling by TurboID in microglial and neuronal cell lines. (31st December 2021)

• Record Type: Journal Article
• Authors: Sunna, Sydney N; Rayaprolu, Sruti; Bowen, Christine A; Bagchi, Pritha; Seyfried, Nicholas T.; Rangaraju, Srikant
• Is Part Of: Alzheimer's & dementia. Volume 17(2021)Supplement 12
• Page Start: n/a
• Page End: n/a
• Publication Date: 2021-12-31
• Subjects: Alzheimer's disease -- Periodicals; Alzheimer Disease -- Periodicals; Dementia -- Periodicals; Démence; Maladie d'Alzheimer; Périodique électronique (Descripteur de forme); Ressource Internet (Descripteur de forme); 616.83
• DOI: 10.1002/alz.058534
• Languages: English
• ISSNs: 1552-5260

Abstract

Abstract: Background: Despite advancements in systems‐level analyses to Alzheimer's disease, global proteomic profiling of brain homogenate cannot resolve molecular changes occurring in distinct cell types. Proximity labeling of proteins by the biotin‐ligase TurboID, coupled with mass spectrometry (MS) analyses of biotinylated proteins, can resolve cell‐type specific proteomes from brain without cell type enrichment. Before in‐vivo applications, it is important to identify proteins preferentially labeled by TurboID in mammalian cells, and directly compare biotinylated proteomes with whole cell proteomes. We determined whether TurboID‐labeled proteomes in microglial (BV2) and neuronal (N2A) cell lines are representative of total cellular proteomes under homeostatic and activated conditions. Method: We generated N2A and BV2 cell lines that stably express TurboID fused to a nuclear export sequence. Cells incubated in 200μM biotin and 1μg/mL lipopolysaccharide (LPS) for 48hrs to mimic inflammatory stress. Western blotting and label‐free quantitation MS identified biotinylated proteins in whole‐cell‐lysates and streptavidin‐enriched preparations. Result: Western‐blot confirmed robust biotinylation in TurboID N2A and BV2 cell‐lines. LFQ‐MS quantified >3, 000 proteins in inputs and >2, 400 proteins in biotinylated proteomes from both cell types. In BV2 and N2A inputs, TurboID expression and biotinylation didn't impact the proteome, evidenced by few differentially‐expressed proteins and absence of cellular respiration measures in mitochondrial stress‐tests, comparing TurboID and non‐TurboID cells. Of all the proteins quantified in BV2 and N2A TurboID inputs, ∼50% were also quantified in the biotinylated proteomes in both cell types. Compared to input proteomes of both cell types, the biotinylated proteome was enriched in vesicular, ribosomal, mitochondrial, cytoplasmic and cytoskeletal proteins. The biotinylated proteome successfully differentiated BV2 and N2A cells. LPS stimulation altered levels of 759 proteins in BV2 cells in the input proteome, of which the top increased (e.g. Irg1, Oasl1, Cyb5a) and decreased (e.g. Mrc1, Mgl2, Arg1) proteins showed concordant changes in the biotinylated BV2 proteome. Conclusion: Our analyses show that proteomic labeling by TurboID in microglial and neuronal cell lines doesn't disrupt cellular function. The resulting biotinylated proteomes are reasonably representative of the whole‐cell proteome and captures the most robust alterations induced by immune stimuli in microglia. These foundational data will guide future applications of proximity‐labeling for cell‐type‐specific proteomics in‐vivo.