Multiplex measurement of protein–peptide dissociation constants using dialysis and mass spectrometry

We propose a high‐throughput method for quantitatively measuring hundreds of protein–peptide binding affinities in parallel. In this assay, a solution of protein is dialyzed into a buffer containing a pool of potential binding peptides, such that upon equilibration the relative abundance of a peptide species is mathematically related to that peptide's dissociation constant, K ( d ). We use isobaric multiplexed quantitative proteomics to simultaneously determine the relative abundance, and hence the K ( d ) and its associated error, for an entire peptide library. We apply this technique, which we call PEDAL (parallel equilibrium dialysis for affinity learning), to determine accurate K ( d )'s between a PDZ domain and hundreds of peptides, spanning an affinity range of multiple orders of magnitude in a single experiment. PEDAL is a convenient, fast, and low‐cost method for measuring large numbers of protein–peptide affinities in parallel, providing a rare combination of true in‐solution binding equilibria with the ability to multiplex.