Mapping Differential Protein-Protein Interaction Networks using Affinity Purification Mass Spectrometry.

Area of Science:

• Proteomics
• Molecular Biology
• Systems Biology

Background:

• Cellular functions rely on protein complexes, whose remodeling by mutations or signaling changes drives health and disease outcomes.
• Understanding dynamic protein-protein interactions (PPIs) is crucial for deciphering cellular mechanisms.
• Existing methods may not fully capture global network alterations between conditions.

Approach:

• Presents a detailed affinity purification-mass spectrometry (APMS) protocol for quantifying and visualizing global changes in PPI networks.
• Covers essential steps from designing affinity-tagged 'bait' proteins in mammalian cells to data analysis and visualization.
• Includes methods for protein complex identification, differential PPI quantification, and network visualization.

Key Points:

• The protocol enables the identification and quantification of differential PPIs between pairwise conditions.
• It details the entire workflow, including gene construct design, cell culture, affinity purification, mass spectrometry, and data analysis.
• Provides a framework for assessing the impact of genetic mutations or cellular signaling on protein interaction networks.

Conclusions:

• This APMS proteomics protocol offers a comprehensive approach to study global changes in protein-protein interaction networks.
• It facilitates the mechanistic understanding of phenotypic outcomes in health and disease driven by protein complex remodeling.
• The protocol's applicability across various cell types and biological areas is discussed, along with its limitations.