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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.

Liu et al. reported a non-radioactive alternative to analyze newly synthesized proteins in cell culture and whole organisms that is based on an alkyne analog of puromycin (Fig. 1). The cell-permeable O-propargyl-puromycin (Fig. 1A) incorporates 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[1,2,3].

In contrast to previously reported non-radioactive methionine analog-approaches[4,5], methionine free-medium is not required for O-propargyl-puromycin-based monitoring of nascent protein synthesis.

Figure 1: O-propargyl-puromycin labels newly synthesized proteins in cell culture and whole organisms (modified according to [1]. A) Chemical structure of O-propargyl-puromycin (OP-Puro). Visualization of incorporated OP-puro is performed via Cu(I)-catalyzed Click Chemistry (CuAAC) with Azides of fluorescent dyes → Click Chemistry or Azides of Biotin → Click Chemistry. B) Nascent protein expression in crypts of mouse small intestine was visualized by whole-mount staining. O-Propargyl-puromycin labeled proteins were detected with Tamra-azide (red) and nuclear DNA was stained with OliGreen (green) (modified according to [1]) C) Newly synthesized proteins in NIH3T3 cells are rapidly detected by incubation with 50 µM OP-puro. OP-puro labeled proteins have been visualized by Alexa568-azide, nuclear DNA was stained with Hoechst dye (modified according to [1]).

Selected References

[1] 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.
[2] Goodman et al. (2012) Imaging of protein synthesis with puromycin. Proc. Natl. Acad. Sci. USA 109 (17):E989.
[3] 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.
[4] Dieterich et al. (2010) In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nature Neuroscience 13 (7):897.
[5] 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.