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Supramolecular fishing for plasma membrane proteins using an ultrastable synthetic host–guest binding pair

Nature Chemistry volume 3, pages 154–159 (2011)Cite this article

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Abstract

Membrane proteomics, the large-scale global analysis of membrane proteins, is often constrained by the efficiency of separating and extracting membrane proteins. Recent approaches involve conjugating membrane proteins with the small molecule biotin and using the receptor streptavidin to extract the labelled proteins. Despite the many advantages of this method, several shortcomings remain, including potential contamination by endogenously biotinylated molecules and interference by streptavidin during analytical stages. Here, we report a supramolecular fishing method for membrane proteins using the synthetic receptor–ligand pair cucurbit[7]uril–1-trimethylammoniomethylferrocene (CB[7]–AFc). CB[7]-conjugated beads selectively capture AFc-labelled proteins from heterogeneous protein mixtures, and AFc-labelling of cells results in the efficient capture of membrane proteins by these beads. The captured proteins can be recovered easily at room temperature by treatment with a strong competitor such as 1,1′-bis(trimethylammoniomethyl)ferrocene. This synthetic but biocompatible host–guest system may be a useful alternative to streptavidin–biotin for membrane proteomics as well as other biological and biotechnological applications.

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Figure 1: Strategy for isolation of plasma membrane proteins using an ultrastable synthetic binding pair system.
Figure 2: Preparation of CB[7] beads and synthesis of AFc–COOH (4), followed by in situ labelling of protein (ferrocenylation).
Figure 3: Isolation of AFc–BSA using CB[7] beads.
Figure 4: Protein capture ability of the CB[7]–AFc and streptavidin–biotin systems.
Figure 5: Total plasma membrane protein isolation using CB[7]–AFc and streptavidin-biotin systems.
Figure 6: Immunoblotting of subcellular marker proteins from Rat-1 cells.
Figure 7: Recovery of isolated total plasma membrane proteins by treatment with free guest (BAFc) as a strong competitor to AFc.

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Acknowledgements

This work was supported by the Creative Research Initiative and Brain Korea 21 Program of the Korean Ministry of Education, Science, and Technology (MOEST), the World Class University (WCU) program through the Korea Science and Engineering Foundation funded by MOEST (project no. R31-2008-000-10059-0). The authors also thank J. Mark Kim for helpful discussions.

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Authors and Affiliations

  1. Department of Chemistry, National Creative Research Initiative Center for Smart Supramolecules(CSS), Pohang University of Science and Technology(POSTECH), Pohang, 790-784, Republic of Korea

    Don-Wook Lee, Kyeng Min Park, Mainak Banerjee, Kyungwon Suh, Somak Paul, Hyuntae Jung, Narayanan Selvapalam & Kimoon Kim

  2. School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology(POSTECH), Pohang, 790-784, Republic of Korea

    Don-Wook Lee, Hyuntae Jung, Sung Ho Ryu & Kimoon Kim

  3. Division of Advanced Materials Science, Pohang University of Science and Technology(POSTECH), Pohang, 790-784, Republic of Korea

    Kyeng Min Park, Narayanan Selvapalam & Kimoon Kim

  4. NovaCell Technology Inc., Pohang, 790-834, Republic of Korea

    Sang Hoon Ha, Taehoon Lee & Jaeyoon Kim

  5. Division of Molecular and Life Sciences, Department of Life Science, Pohang University of Science and Technology(POSTECH), Pohang, 790-784, Republic of Korea

    Sung Ho Ryu

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  1. Don-Wook Lee
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Contributions

K.K. and S.H.R conceived and designed the project. D.W.L., K.M.P., B.M., K.S., P.S. and N.S. performed the synthesis. D.W.L. and H.J. performed the protein and cell experiments. D.W.L., K.M.P. and S.H.H. analysed the data. T.L. performed mass spectrometry. J.K. provided invaluable advice. K.K., D.W.L. and K.M.P. wrote the paper.

Corresponding authors

Correspondence to Sung Ho Ryu or Kimoon Kim.

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The authors declare no competing financial interests.

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Lee, DW., Park, K., Banerjee, M. et al. Supramolecular fishing for plasma membrane proteins using an ultrastable synthetic host–guest binding pair. Nature Chem 3, 154–159 (2011). https://doi.org/10.1038/nchem.928

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