Mapping protein interactions by combining antibody affinity maturation and mass spectrometry

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• Antibody generation for immunoprecipitation is often limited by antibody affinity and epitope accessibility.
• Mapping protein interactions is crucial for understanding cellular processes.

Purpose of the Study:

• To develop a scalable method for generating high-affinity antibodies for immunoprecipitation.
• To improve the efficiency of mapping protein-protein interaction networks.

Main Methods:

• Phage display and affinity maturation were used to generate and optimize antibodies.
• Antibody variable heavy (V(H)) and variable light (V(L)) genes were combined through chain shuffling.
• A 96-well immunocapture screen was developed to predict antibody performance.
• Mass spectrometry was used to identify protein binding partners and phosphorylation sites.

Main Results:

• Chain shuffling generated improved antibody binders with higher affinity.
• A clear affinity threshold (60 nM for SHC1) was identified for successful immunoprecipitation.
• The method successfully identified known SHC1 binding partners and phosphorylation sites in EGF-stimulated breast cancer cells.

Conclusions:

• Antibody chain shuffling is a scalable approach for generating immunoprecipitation-capable antibodies.
• This method enhances the ability to map protein-protein interaction networks.
• Affinity maturation is critical for successful antibody-based protein interaction studies.