title: Metabolic labeling with noncanonical amino acids and visualization by chemoselective fluorescent tagging.
abstract: 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.
authors.affiliation: Max Planck Institute for Brain Research, Department of Synaptic Plasticity, Frankfurt, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Tom Dieck
authors.affiliation: Leibniz Institute for Neurobiology, Research Group Neuralomics, Magdeburg, Germany.|Otto-von-Guericke-University Magdeburg, Medical Faculty, Institute for Pharmacology and Toxicology, Magdeburg, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Müller
authors.affiliation: Max Planck Institute for Brain Research, Department of Synaptic Plasticity, Frankfurt, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Nehring
authors.affiliation: Max Planck Institute for Brain Research, Department of Synaptic Plasticity, Frankfurt, Germany.
authors.forename: Flora I
authors.initials: FI
authors.lastname: Hinz
authors.affiliation: Max Planck Institute for Brain Research, Department of Synaptic Plasticity, Frankfurt, Germany.
authors.forename: Ina
authors.initials: I
authors.lastname: Bartnik
authors.affiliation: Max Planck Institute for Brain Research, Department of Synaptic Plasticity, Frankfurt, Germany.
authors.forename: Erin M
authors.initials: EM
authors.lastname: Schuman
authors.affiliation: Leibniz Institute for Neurobiology, Research Group Neuralomics, Magdeburg, Germany.|Otto-von-Guericke-University Magdeburg, Medical Faculty, Institute for Pharmacology and Toxicology, Magdeburg, Germany.
authors.forename: Daniela C
authors.initials: DC
authors.lastname: Dieterich
mesh_terms: D000818:Animals; D001386:Azides / Q000737:chemistry* / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008715:Methionine / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001386:Azides; D005456:Fluorescent Dyes; D011506:Proteins; D008715:Methionine