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

ABSTRACT: 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 mu

SUBMITTER: Zhao Y 

PROVIDER: S-EPMC10031237 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature