RESUMEN
Fluorescent labeling of proteins by genetically encoded fluorescent protein tags has enabled an enhanced understanding of cell biological processes but is restricted to the analysis of a limited number of identified proteins. This approach does not permit, e.g., the unbiased visualization of a full proteome in situ. We describe here a fluorescence-based method to follow proteome-wide patterns of newly synthesized proteins in cultured cells, tissue slices, and a whole organism. This technique is compatible with immunohistochemistry and in situ hybridization. Key to this method is the introduction of a small bio-orthogonal reactive group by metabolic labeling. This is accomplished by replacing the amino acid methionine by the azide-bearing methionine surrogate azidohomoalanine (AHA) in a step very similar to classical radioisotope labeling. Subsequently, an alkyne-bearing fluorophore is covalently attached to the group by "click chemistry"--a copper(I)-catalyzed [3+2]azide-alkyne cycloaddition. By similar means, metabolic labeling can also be performed with the alkyne-bearing homopropargylglycine (HPG) and clicked to an azide-functionalized fluorophore.
Asunto(s)
Azidas/química , Técnicas de Cultivo de Célula/métodos , Colorantes Fluorescentes/química , Inmunohistoquímica/métodos , Hibridación Fluorescente in Situ/métodos , Metionina/metabolismo , Proteínas/química , Animales , Azidas/metabolismo , Colorantes Fluorescentes/metabolismo , Humanos , Metionina/química , Proteínas/metabolismoRESUMEN
Local protein synthesis and its activity-dependent modulation via dopamine receptor stimulation play an important role in synaptic plasticity - allowing synapses to respond dynamically to changes in their activity patterns. We describe here the metabolic labeling, enrichment, and MS-based identification of candidate proteins specifically translated in intact hippocampal neuropil sections upon treatment with the selective D1/D5 receptor agonist SKF81297. Using the noncanonical amino acid azidohomoalanine and click chemistry, we identified over 300 newly synthesized proteins specific to dendrites and axons. Candidates specific for the SKF81297-treated samples were predominantly involved in protein synthesis and synapse-specific functions. Furthermore, we demonstrate a dendrite-specific increase in proteins synthesis upon application of SKF81297. This study provides the first snapshot in the dynamics of the dopaminergic hippocampal neuropil proteome.