BONCAT: Metabolic Labeling, Click Chemistry, and Affinity Purification of Newly Synthesized Proteomes
Metabolic labeling of proteins using classical radioisotope-labeled amino acids has enabled the analysis and function of protein synthesis for many biological processes but cannot be combined with modern high-throughput mass spectrometry analysis. This chapter describes the unbiased identification of a whole de novo synthesized proteome of cultured cells or of a translationally active subcellular fraction of the mammalian brain. This technique relies on the introduction of a small bioorthogonal reactive group by metabolic labeling accomplished by replacing the amino acid methionine by the azide-bearing methionine surrogate azidohomoalanine (AHA) or the amino acid homopropargylglycine (HPG). Subsequently an alkyne- or azide-bearing affinity tag is covalently attached to the group by “click chemistry”—a copper(I)-catalyzed [3+2] azide-alkyne cycloaddition. Affinity tag-labeled proteins can be analyzed in candidate-based approaches by conventional biochemical methods or with high-throughput mass spectrometry.
Key wordsProtein synthesis Click chemistry Affinity purification Proteome Mass spectrometry
This work has received funding from the Deutsche Forschungsgemeinschaft (DI1512/1-1 and DI1512/1-2), the DIP (Deutsch-Israelische-Projektkooperation) German-Israeli Project Cooperation foundation, and the CBBS, Magdeburg, Germany, to DCD.
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