title: Monitoring Astrocytic Proteome Dynamics by Cell Type-Specific Protein Labeling.
abstract: The ability of the nervous system to undergo long-term plasticity is based on changes in cellular and synaptic proteomes. While many studies have explored dynamic alterations in neuronal proteomes during plasticity, there has been less attention paid to the astrocytic counterpart. Indeed, progress in identifying cell type-specific proteomes is limited owing to technical difficulties. Here, we present a cell type-specific metabolic tagging technique for a mammalian coculture model based on the bioorthogonal amino acid azidonorleucine and the mutated Mus musculus methionyl-tRNA synthetaseL274G enabling azidonorleucine introduction into de novo synthesized proteins. Azidonorleucine incorporation resulted in cell type-specific protein labeling and retained neuronal or astrocytic cell viability. Furthermore, we were able to label astrocytic de novo synthesized proteins and identified both Connexin-43 and 60S ribosomal protein L10a upregulated upon treatment with Brain-derived neurotrophic factor in astrocytes of a neuron-glia coculture. Taken together, we demonstrate the successful dissociation of astrocytic from neuronal proteomes by cell type-specific metabolic labeling offering new possibilities for the analyses of cell type-specific proteome dynamics.
authors.affiliation: Neural Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Müller
authors.affiliation: Neural Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Stellmacher
authors.affiliation: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Freitag
authors.affiliation: Neural Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Landgraf
authors.affiliation: Neural Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany.|Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany.|Center for Behavioral Brain Sciences, Magdeburg, Germany.
authors.forename: Daniela C
authors.initials: DC
authors.lastname: Dieterich
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D001386:Azides / Q000032:analysis / Q000737:chemistry; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D018920:Coculture Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008718:Methionine-tRNA Ligase / Q000235:genetics / Q000378:metabolism; D009457:Neuroglia / Q000166:cytology; D009646:Norleucine / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D020543:Proteome; D040901:Proteomics / Q000379:methods; D017208:Rats, Wistar
chemical_list: D001386:Azides; D019208:Brain-Derived Neurotrophic Factor; D011506:Proteins; D020543:Proteome; C506098:azidohomoalanine; C000609218:azidonorleucine; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D009646:Norleucine; D008718:Methionine-tRNA Ligase; D000409:Alanine