title: 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 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.
authors.affiliation: Institute of Pharmacology and Toxicology, Otto-von-Guericke University Magdeburg, 39120, Magdeburg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Landgraf
authors.forename: Elmer R
authors.initials: ER
authors.lastname: Antileo
authors.forename: Erin M
authors.initials: EM
authors.lastname: Schuman
authors.forename: Daniela C
authors.initials: DC
authors.lastname: Dieterich
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity; D057930:Click Chemistry; D061565:Cycloaddition Reaction; D057809:HEK293 Cells; D006801:Humans; D020543:Proteome / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D013194:Staining and Labeling
chemical_list: D020543:Proteome; C506098:azidohomoalanine; D000409:Alanine