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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.
