µMap Photoproximity Labeling on the Cell Surface

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.