BONCAT: metabolic labeling, click chemistry, and affinity purification of newly synthesized proteomes

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1266; p. 199
• Main Authors: Landgraf, Peter, Antileo, Elmer R, Schuman, Erin M, Dieterich, Daniela C
• Format: Journal Article
• Language: English
• Published: United States 2015
• Subjects: Alanine - analogs & derivatives; Alanine - chemistry; Alanine - metabolism; Chromatography, Affinity; Click Chemistry; Cycloaddition Reaction; HEK293 Cells; Humans; Proteome - biosynthesis; Proteome - chemistry; Proteome - isolation & purification; Staining and Labeling
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-2272-7_14

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.