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

Abstract

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 highthroughput mass spectrometry analysis. This chapter describes the unbiased identifi cation 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 affi nity 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 candidatebased approaches by conventional biochemical methods or with high-throughput mass spectrometry.