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

TL;DR

This paper introduces a comprehensive APMS proteomics protocol for quantifying and visualizing changes in protein-protein interaction networks across different conditions, aiding understanding of cellular functions and disease mechanisms.

Contribution

It provides a detailed, step-by-step protocol for mapping differential PPIs using affinity purification mass spectrometry, including data analysis and visualization methods.

Findings

Effective identification of differential PPIs across conditions.

Robust visualization of PPI network changes.

Protocol applicable across various cell types and biological contexts.

Abstract

Proteins congregate into complexes to perform fundamental cellular functions. Phenotypic outcomes, in health and disease, are often mechanistically driven by the remodeling of protein complexes by protein coding mutations or cellular signaling changes in response to molecular cues. Here, we present an affinity purification mass spectrometry (APMS) proteomics protocol to quantify and visualize global changes in protein protein interaction (PPI) networks between pairwise conditions. We describe steps for expressing affinity tagged bait proteins in mammalian cells, identifying purified protein complexes, quantifying differential PPIs, and visualizing differential PPI networks. Specifically, this protocol details steps for designing affinity tagged bait gene constructs, transfection, affinity purification, mass spectrometry sample preparation, data acquisition, database search, data quality control, PPI confidence scoring, cross run normalization, statistical data analysis, and differential PPI visualization. Our protocol discusses caveats and limitations with applicability across cell types and biological areas.