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BONCAT: Metabolic Labeling, Click Chemistry, and Affinity Purification of Newly Synthesized Proteomes

  • Peter Landgraf
  • Elmer R. Antileo
  • Erin M. SchumanEmail author
  • Daniela C. DieterichEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1266)

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.

Key words

Protein synthesis Click chemistry Affinity purification Proteome Mass spectrometry 

Notes

Acknowledgments

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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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Institute of Pharmacology and ToxicologyOtto-von-Guericke University MagdeburgMagdeburgGermany
  2. 2.Emmy Noether Group NeuralomicsLeibniz Institute for NeurobiologyMagdeburgGermany
  3. 3.Max Planck Institute for Brain ResearchFrankfurt am MainGermany

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