Studying the spatial distribution of proteomes in cells is of great significance for elucidating many molecular mechanisms in cell biology. In recent years, proximity chemical labeling technology represented by APEX (The engineered ascorbate peroxidase) has developed rapidly, achieving proximity labeling of proteins and RNA in specific spatial regions within a living cell environment, avoiding false positives caused by traditional physical separation of organelles.

APEX technology uses peroxidase to catalyze the probe substrate biotin-phenol (BP) to generate highly active free radical intermediates, which can perform covalent addition reactions on surrounding proteins and RNA to achieve spatial specific labeling [1]. APEX can be applied to multiple biological systems [2-5], but the large molecular size and low solubility of traditional BP substrates limit its application in the fields of yeast, bacteria and other microorganisms.

The research group of Researcher Zou Peng of Peking University designed and successfully synthesized a new generation of APEX probe substrate for microbial systems - Alkyne-Phenol (Alk-Ph) [6].

Compared with traditional substrate BP, it has the following advantages:
Smaller molecular size (MW: 217.27);
Better cell wall and cell membrane penetration, higher solubility in aqueous solutions;
Good spatial specificity and higher coverage increase APEX labeling efficiency by an order of magnitude (Figure 1).

Researcher Zou Peng's research group has successfully used Alk-Ph to proximity label proteins (Figure 2) and RNA (Figure 3) in living yeast cells, and used quantitative proteomic and transcriptomic data to prove that the probe has good Spatial specificity and higher coverage.

Alk-Ph probes can also reveal APEX modification sites on proteins, providing technical support for in-depth study of protein topology and conformation.

References

Rhee, H.-W., Zou, P., Udeshi, N.D., Martell, J.D., Mootha, V.K., Carr, S.A., and Ting, A.Y. (2013). Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging. Science 339, 1328-1331.
Hung, V., Lam, S.S., Udeshi, N.D., Svinkina, T., Guzman, G., Mootha, V.K., Carr, S.A., and Ting, A.Y. (2017). Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation. eLife 6, e24463.
Hung, V., Udeshi, N.D., Lam, S.S., Loh, K.H., Cox, K.J., Pedram, K., Carr, S.A., and Ting, A.Y. (2016). Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat Protoc 11, 456-475.
Hung, V., Zou, P., Rhee, H.-W., Udeshi, Namrata D., Cracan, V., Svinkina, T., Carr, Steven A., Mootha, Vamsi K., and Ting, Alice Y. (2014). Proteomic Mapping of the Human Mitochondrial Intermembrane Space in Live Cells via Ratiometric APEX Tagging. Mol Cell 55, 332-341.
Paek, J., Kalocsay, M., Staus, D.P., Wingler, L., Pascolutti, R., Paulo, J.A., Gygi, S.P., and Kruse, A.C. (2017). Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling. Cell 169, 338-349.
Li, Y., Tian, C., Liu, K., Zhou, Y., Yang, J., and Zou, P. (2020). A Clickable APEX Probe for Proximity-Dependent Proteomic Profiling in Yeast. Cell Chem Biol 27, 858-865.e858.