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Related Experiment Video

Updated: Jan 6, 2026

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT
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µMap Photoproximity Labeling on the Cell Surface.

Hong Kai Ng1,2,3, Cameron J Douglas1,2,3, Ciaran P Seath1

  • 1Department of Chemistry, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, Jupiter, Florida.

Current Protocols
|October 8, 2025
PubMed
Summary

Antibody-guided µMap photoproximity labeling maps protein interactions near cell surfaces. This method precisely identifies the interactome of HER2 (Human Epidermal growth factor Receptor 2) without genetic modification.

Keywords:
cellular membraneinteractomephotochemistryproteomicsproximity labeling

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Proteomics

Background:

  • Understanding protein interactions is key to deciphering cellular functions and disease mechanisms.
  • Existing methods like co-immunoprecipitation struggle with membrane-bound proteins.
  • Enzyme-based proximity labeling has limitations in labeling radius, cellular engineering needs, and amino acid biases.

Purpose of the Study:

  • To present antibody-guided µMap photoproximity labeling as a superior method for in situ interactome mapping.
  • To detail protocols for preparing iridium-antibody conjugates and applying them for mass spectrometry-based interactome analysis.
  • To demonstrate the method's efficacy using HER2 (Human Epidermal growth factor Receptor 2) as a model cell surface protein.

Main Methods:

  • Utilized antibody-guided µMap photoproximity labeling targeting endogenous HER2.
  • Developed protocols for preparing and validating iridium-antibody conjugates.
  • Applied proximity labeling followed by streptavidin enrichment for mass spectrometry analysis.

Main Results:

  • Successfully mapped the interactome of HER2 using antibody-guided µMap labeling.
  • Demonstrated the method's ability to work without cellular engineering.
  • Leveraged µMap's short 4-nm labeling radius for precise in situ interactome analysis.

Conclusions:

  • Antibody-guided µMap photoproximity labeling overcomes limitations of previous interactome mapping techniques.
  • The described protocols are effective for studying cell surface protein interactomes.
  • This broadly applicable method facilitates the study of any cell surface protein using available antibodies.