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

Background

Bio-orthogonal chemical reporters are used to visualize and identify experimentally defined entities of macromolecules in living cells and organisms. Originally developed as a means for protein tracking and thus complementary to tagging by fluorescent proteins (e.g., GFP) or antibody-labeling (Tsien, 1998; Massoud and Gambhir, 2003), they can also be used to monitor glycans, lipids, nucleic acids or virus particles (Wang et al., 2003; Kho et al., 2004; Seo et al., 2004; Laughlin et al., 2008; Martin et al., 2008; Martin and Cravatt, 2009; Palaniappan and Bertozzi, 2016, Nguyen et al., 2017). Azides and alkynes are particularly well suited as bio-orthogonal chemical reporters (Prescher and Bertozzi, 2005; Dieterich, 2010) because they typically do not exist in living systems, thus precluding unwanted side reactions during tagging. At the same time, and in contrast to radioactive reporters, they allow for highly specific reactions with established tags (e.g., fluorophores or affinity tags).

The traditional copper-catalyzed azide-alkyne-cycloaddition (CuAAC [Rostovtsev et al., 2002; Tornoe et al., 2002], AAC [Huisgen, 1963]), widely known as ‘click chemistry’ is one of the principal bio-orthogonal labeling reactions as it is fast, chemically highly selective, and applicable over a wide range of biochemically compatible buffers, pH and temperature. Azide groups and alkyne groups exhibit high reactivity towards each other, and covalent bonding between them manifests in the formation of a stable triazole ring. Azide-bearing molecules can be detected using alkyne-bearing tags and vice versa. Whereas, however, alkynes can only be CuAAC-clicked, azides can also be tagged by Staudinger ligation (Saxon and Bertozzi, 2000; Kiick et al., 2002) or strain-promoted cycloaddition (e.g., Agard et al., 2004; reviewed in Lallana et al., 2011), both with a slower reaction rate (to review chemical reactions in bio-orthogonal chemistry see Prescher and Bertozzi, (2005); Ramil and Lin, (2013).

In order to tag proteomes on the basis of click chemistry, we developed bio-orthogonal non-canonical amino acid tagging (BONCAT). In the prototypic, non-cell-type- specific BONCAT approach, the non-canonical amino acid azidohomoalanine (AHA) (or homopropargylglycine [HPG]) is used to compete with methionine for tRNA loading and translational incorporation using the endogenous protein synthesis machinery. AHA bears an azide ‘handle’ for alkyne-bearing biotin tagging, allowing for subsequent protein purification, Western blot or mass spectrometry analyses (Dieterich et al., 2006). Similarly, in fluorescent non-canonical amino acid tagging (FUNCAT), AHA is clicked to an alkyne-bearing fluorophore for immunohistochemistry (Beatty et al., 2006; Dieterich et al., 2010). Most recently, this method was further advanced to enable the direct visualization of single newly synthesized proteins via proximity ligation assay (PLA) techniques (FUNCAT-PLA, tom Dieck et al., 2015). The expansion of the method to cell-type specific labeling with e.g., azidonorleucine (ANL) requires a mutation of the target organism to provide a cell-type specific MetRS capable to recognize ANL (first mutant MetRS capable to process ANL from E. Coli, Link et al., 2006, mMetRS^L274G in astrocytes, Müller et al., 2015; mMetRS^L274G and/or dMetRS^L262G in several Drosophila tissues (Erdmann et al., 2015; Niehues et al., 2015). ANL has a longer side chain than AHA and, therefore, does not fit the binding pocket of the endogenous MetRS. Critically, this approach does not require methionine depletion as the MetRS^LtoG has a higher affinity for ANL compared to AHA.

In the following, we provide detailed BONCAT, and FUNCAT protocols for cell-type specific metabolic labeling of de novo synthesized proteins with ANL in Drosophila according to Erdmann et al. (2015) and Niehues et al. (2015).