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Cellular Proteomic Profiling Using Proximity Labeling by TurboID-NES in Microglial and Neuronal Cell Lines.
Sunna, Sydney; Bowen, Christine; Zeng, Hollis; Rayaprolu, Sruti; Kumar, Prateek; Bagchi, Pritha; Dammer, Eric B; Guo, Qi; Duong, Duc M; Bitarafan, Sara; Natu, Aditya; Wood, Levi; Seyfried, Nicholas T; Rangaraju, Srikant.
Afiliación
  • Sunna S; Department of Neurology, Emory University, Atlanta Georgia, USA; Center for Neurodegenerative Diseases, Emory University, Atlanta, Georgia, USA.
  • Bowen C; Department of Neurology, Emory University, Atlanta Georgia, USA; Center for Neurodegenerative Diseases, Emory University, Atlanta, Georgia, USA; Department of Biochemistry, Emory University, Atlanta, Georgia, USA.
  • Zeng H; Department of Neurology, Emory University, Atlanta Georgia, USA; Center for Neurodegenerative Diseases, Emory University, Atlanta, Georgia, USA.
  • Rayaprolu S; Department of Neurology, Emory University, Atlanta Georgia, USA; Center for Neurodegenerative Diseases, Emory University, Atlanta, Georgia, USA.
  • Kumar P; Department of Neurology, Emory University, Atlanta Georgia, USA; Center for Neurodegenerative Diseases, Emory University, Atlanta, Georgia, USA.
  • Bagchi P; Center for Neurodegenerative Diseases, Emory University, Atlanta, Georgia, USA; Department of Biochemistry, Emory University, Atlanta, Georgia, USA; Emory Integrated Proteomics Core, Emory University, Atlanta, Georgia, USA.
  • Dammer EB; Center for Neurodegenerative Diseases, Emory University, Atlanta, Georgia, USA; Department of Biochemistry, Emory University, Atlanta, Georgia, USA; Emory Integrated Proteomics Core, Emory University, Atlanta, Georgia, USA.
  • Guo Q; Center for Neurodegenerative Diseases, Emory University, Atlanta, Georgia, USA; Department of Biochemistry, Emory University, Atlanta, Georgia, USA; Emory Integrated Proteomics Core, Emory University, Atlanta, Georgia, USA.
  • Duong DM; Center for Neurodegenerative Diseases, Emory University, Atlanta, Georgia, USA; Department of Biochemistry, Emory University, Atlanta, Georgia, USA; Emory Integrated Proteomics Core, Emory University, Atlanta, Georgia, USA.
  • Bitarafan S; George W. Woodruff School of Mechanical Engineering, Wallace H. Coulter Department of Biomedical Engineering, and Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, USA.
  • Natu A; Department of Neurology, Emory University, Atlanta Georgia, USA; Center for Neurodegenerative Diseases, Emory University, Atlanta, Georgia, USA.
  • Wood L; George W. Woodruff School of Mechanical Engineering, Wallace H. Coulter Department of Biomedical Engineering, and Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, USA.
  • Seyfried NT; Department of Neurology, Emory University, Atlanta Georgia, USA; Center for Neurodegenerative Diseases, Emory University, Atlanta, Georgia, USA; Department of Biochemistry, Emory University, Atlanta, Georgia, USA; Emory Integrated Proteomics Core, Emory University, Atlanta, Georgia, USA. Electronic
  • Rangaraju S; Department of Neurology, Emory University, Atlanta Georgia, USA; Center for Neurodegenerative Diseases, Emory University, Atlanta, Georgia, USA. Electronic address: srikant.rangaraju@emory.edu.
Mol Cell Proteomics ; 22(6): 100546, 2023 Jun.
Article en En | MEDLINE | ID: mdl-37061046
ABSTRACT
Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific approaches at the protein (proteomic) level can provide biological and therapeutic insights. To overcome the barriers of conventional isolation-based methods for cell type-specific proteomics, in vivo proteomic labeling with proximity-dependent biotinylation of cytosolic proteins using biotin ligase TurboID, coupled with mass spectrometry (MS) of labeled proteins, emerged as a powerful strategy for cell type-specific proteomics in the native state of cells without the need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID approach are representative of the whole-cell proteome and capture cellular responses to stimuli without disruption of cellular processes. To address this, we generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing cytosolic TurboID to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under homeostatic conditions. TurboID labeled endolysosome, translation, vesicle, and signaling proteins in BV2 microglia and synaptic, neuron projection, and microtubule proteins in N2A neurons. TurboID expression and biotinylation minimally impacted homeostatic cellular proteomes of BV2 and N2A cells and did not affect lipopolysaccharide-mediated cytokine production or resting cellular respiration in BV2 cells. MS analysis of the microglial biotin-labeled proteins captured the impact of lipopolysaccharide treatment (>500 differentially abundant proteins) including increased canonical proinflammatory proteins (Il1a, Irg1, and Oasl1) and decreased anti-inflammatory proteins (Arg1 and Mgl2).
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Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Microglía / Proteoma Límite: Animals Idioma: En Revista: Mol Cell Proteomics Asunto de la revista: BIOLOGIA MOLECULAR / BIOQUIMICA Año: 2023 Tipo del documento: Article País de afiliación: Estados Unidos
Texto completo
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Microglía / Proteoma Límite: Animals Idioma: En Revista: Mol Cell Proteomics Asunto de la revista: BIOLOGIA MOLECULAR / BIOQUIMICA Año: 2023 Tipo del documento: Article País de afiliación: Estados Unidos