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

The accurate description and subsequent modeling of protein interactomes require quantification of their affinities at the proteome-wide scale. Here we develop and validate the Holdup Multiplex, a versatile assay with a mass spectrometry (MS) readout for profiling the affinities of a protein for large pools of peptides. The method can precisely quantify, in one single run, thousands of affinity constants over several orders of magnitude. The throughput, dynamic range, and sensitivity can be pushed to the performance limit of the MS readout. We applied the Holdup Multiplex to quantify in a few sample runs the affinities of the 14-3-3s, phosphoreader proteins highly abundant in humans, for 1000 different phosphopeptides. The seven human 14-3-3 isoforms were found to display similar specificities but staggered affinities, with 14-3-3γ being always the best binder and 14-3-3ε and σ being the weakest. Hundreds of new 14-3-3 binding sites were identified. We also identified dozens of 14-3-3 binding sites, some intervening in key signaling pathways, that were either stabilized or destabilized by the phytotoxin Fusicoccin-A. The results were corroborated by X-ray crystallography. Finally, we demonstrated the transferability of the Holdup Multiplex by quantifying the interactions of a PDZ domain for 5400 PBM peptides at once. The approach is applicable to any category of protein-binding ligands that can be quantifiable by mass spectrometry.