The Holdup Multiplex, an assay for high-throughput measurement of protein-ligand affinity constants using a mass-spectrometry readout

We developed the Holdup Multiplex, which quantifies hundreds of protein-peptide affinities at unprecedented accuracy with only one single experiment, using a label-free quantitative mass-spectrometry strategy. We applied this strategy to the seven human 14 3 3 proteins, and depicted new rules for 14 3 3 binding and specificity, valid at the proteome scale. Our approach is readily transferable to affinity-quantify the interactome of any protein of interest for any pool of ligands.