Puromycin-based Fluorescent & Biotin Protein Labeling

The dynamics of global protein synthesis (both spatial and temporal) is an essential parameter to characterize the cellular response under various physiological and pathological conditions. Current studies of the cellular protein level rely on indirect methods such as DNA and mRNA microarrays or classical radioactive metabolic labeling with ³⁵S-methionine.^[1]

Liu et al. and Gee et al. reported a non-radioactive alternative to analyze newly synthesized proteins that is based on an Alkyne- or Azide-modified puromycin analog, respectively. The cell-permeable O-propargyl-puromycin and 5'-Azido-puromycin incorporate into the C-terminus of translating polypeptide chains thereby stopping translation.

The resulting C-terminal Alkyne-labeled proteins can be detected via Cu(I)-catalyzed click chemistry that offers the choice to introduce a Biotin group (Azides of Biotin → Click Chemistry) for subsequent purification tasks or a fluorescent group (Azides of fluorescent dyes → Click Chemistry for subsequent microscopic imaging^[2,3,4]. The resulting C-terminal Azide-labeled proteins can be detected via copper-free click chemistry using DBCO-modified Biotin → Click Chemistry or DBCO-modified fluorescent dyes → Click Chemistry for subsequent microscopic imaging^[5].

In contrast to previously reported non-radioactive methionine analog-approaches^[6,7], methionine free-medium is not required for O-propargyl-puromycin- and 5'-Azido-puromycin-based monitoring of nascent protein synthesis.

Selected References

[1] Tang et al. (2023) Nascent Proteomics: Chemical Tools for Monitoring Newly Synthesized Proteins. Angew. Chem. Int.Ed.62:e202305866 (1-11).
[2] Liu et al. (2012) Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin. Proc. Natl. Acad. Sci. USA 109 (2):413.
[3] Goodman et al. (2012) Imaging of protein synthesis with puromycin. Proc. Natl. Acad. Sci. USA 109 (17):E989.
[4] Salic et al. (2012) Reply to Goodman et al.: Imaging protein synthesis with puromycin and the subcellular localization of puromycin-polypeptide conjugates. Proc. Natl. Acad. Sci. USA 109 (17):E990.
[5] Gee et al. (2016) Puromycin Analogues Capable of Multiplexed Imaging and Profiling of Protein Synthesis and Dynamics in Live Cells and Neurons. Angew. Chem. Int.Ed.55:4933.
[6] Dieterich et al. (2010) In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nature Neuroscience 13 (7):897.
[7] Dieck et al. (2012) Metabolic Labeling with Noncanonical Amino Acids and Visualisation by Chemoselective Fluorescent Tagging. Current Protocols in Cell Biology 7:7.11.1.