Click Chemistry (CuAAC) and Detection of Tagged de novo Synthesized Proteins in Drosophila

Kathrin Marter; Oliver Kobler; Ines Erdmann; Elaheh Soleimanpour; Peter Landgraf; Anke Müller; Julia Abele; Ulrich Thomas; Daniela C Dieterich

doi:10.21769/BioProtoc.3142

Click Chemistry (CuAAC) and Detection of Tagged de novo Synthesized Proteins in Drosophila

Bio Protoc. 2019 Jan 20;9(2):e3142. doi: 10.21769/BioProtoc.3142.

Authors

Kathrin Marter^{1

2}, Oliver Kobler³, Ines Erdmann^{1

2}, Elaheh Soleimanpour¹, Peter Landgraf^{1

2}, Anke Müller^{1

2}, Julia Abele^{1

2}, Ulrich Thomas³, Daniela C Dieterich^{1

2

4}

Affiliations

¹ Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany.
² Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany.
³ Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany.
⁴ Center for Behavioral Brain Sciences, Magdeburg, Germany.

Abstract

Copper-catalyzed azide-alkyne-cycloaddition (CuAAC), also known as 'click chemistry' serves as a technique for bio-orthogonal, that is, bio-compatible labeling of macromolecules including proteins or lipids. Click chemistry has been widely used to covalently, selectively, and efficiently attach probes such as fluorophores or biotin to small bio-orthogonal chemical reporter groups introduced into macromolecules. In bio-orthogonal non-canonical amino acid tagging (BONCAT) and fluorescent non-canonical amino acid tagging (FUNCAT) proteins are metabolically labeled with a non-canonical, azide-bearing amino acid and subsequently CuAAC-clicked either to an alkyne-bearing biotin (BONCAT) for protein purification, Western blot, or mass spectrometry analyses or to an alkyne-bearing fluorophore (FUNCAT) for immunohistochemistry. In combination with mass spectrometry, these kinds of labeling and tagging strategies are a suitable option to identify and characterize specific proteomes in living organisms without the need of prior cell sorting. Here, we provide detailed protocols for FUNCAT and BONCAT click chemistry and the detection of tagged de novo synthesized proteins in Drosophila melanogaster.

Keywords: Bio-orthogonal chemical reporters; Click chemistry; CuAAC; Drosophila melanogaster; Protein synthesis; Protein tagging; Proteomic profiling.