Protein labeling refers to the use of appropriate molecular labels to monitor biological processes, and detect or purify the labeled protein and its binding partners. At present, there are multiple protein labeling techniques such as stable isotope labeling techniques and fluorescent labeling techniques. Among them, fluorescent labeling techniques for proteins are necessary for the analysis of biological phenomena. However, there have following problems need to be considered carefully: (1) precise stoichiometric control in living cells is difficult; (2) the bulkiness of fluorescent tags limits the analysis of inherent physical and biological properties of the proteins. Therefore, novel techniques to specifically and stoichiometrically label intrinsic proteins in living cells should be developed.
Click chemistry is the most promising approach to overcome these challenges, because it has the following advantages[1]: (1) bioorthogonal reactions; (2) mild reaction conditions suitable for fragile biomolecules, cells, and tissues; (3) extremely high reaction ratio; (4) small size of the functional groups for the cross-coupling reactions; and (6) simple metabolic labeling procedures in living cells.
Applications
- Protein labeling by CuAAC click reaction
The copper-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction has been used extensively for the conjugation, immobilization, and purification of biomolecules. Nevertheless, it is used much less in the cellular context due to the toxicity caused by Cu+. Uttamapinant and co-workers introduced copper-chelating ligands (BTTAA or THPTA) into the CuAAC click reaction (Scheme 1), which led to the rate acceleration of CuAAC click reaction and the reduction of cell toxicity. Thus, this reaction can be used for specific protein labeling on living cells and the signal at a rate 25-fold higher than conventional CuAAC click reaction without copper-chelating ligands[2].
- Protein labeling by SPAAC and SPIEDAC click reaction
SPAAC click reaction is also a powerful tool for protein labeling. Nikic et al[3]. described a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) click reaction and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) click reaction, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. This research offers great potential for studying protein dynamics both in vitro and in vivo, as well as for cell biology studies.
References
- Horisawa, K. Specific and quantitative labeling of biomolecules using click chemistry. Frontiers in Physiology.2014, 5: 457.
- Uttamapinant, C.; et al. Fast, cell-compatible click chemistry with copper-chelating azides for biomolecular labeling. Angewandte Chemie. 2012, 124: 1-6.
- Nikic, I.; et al. Labeling proteins on live mammalian cells using click chemistry. Nature Protocols. 2015, 5(10): 780.
