Holdup Multiplex Assay for High-Throughput Measurement of Protein-Ligand Affinity Constants Using a Mass Spectrometry...

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Accurate modeling of protein interactomes necessitates quantifying protein-ligand affinities across the proteome.
• Existing methods face limitations in throughput and dynamic range for large-scale affinity profiling.

Purpose of the Study:

• To develop and validate the Holdup Multiplex, a mass spectrometry-based assay for high-throughput, quantitative profiling of protein-ligand affinities.
• To apply the assay to study the affinities of human 14-3-3 proteins for phosphopeptides and investigate ligand-induced binding site modulation.

Main Methods:

• Development of the Holdup Multiplex assay utilizing mass spectrometry (MS) for affinity quantification.
• Profiling affinities of seven human 14-3-3 isoforms against 1000 phosphopeptides.
• Validation using X-ray crystallography and transferability demonstration with a PDZ domain.

Main Results:

• The Holdup Multiplex precisely quantifies thousands of affinity constants in a single run with high throughput, dynamic range, and sensitivity.
• Identified staggered affinities among 14-3-3 isoforms, with 14-3-3γ as the strongest binder and 14-3-3ε/σ as the weakest.
• Discovered hundreds of novel 14-3-3 binding sites and identified modulation of dozens of sites by Fusicoccin-A, impacting key signaling pathways.

Conclusions:

• The Holdup Multiplex is a versatile and powerful tool for quantitative proteome-wide affinity profiling.
• The study provides new insights into 14-3-3 protein interactions and their regulation.
• The assay is broadly applicable to various protein-ligand interactions quantifiable by mass spectrometry.