Improved Rapid Equilibrium\nDialysis-Mass Spectrometry\n(RED-MS) Method for Measuring Small Molecule–Protein Complex\nBinding Affinities in Solution

Rapid equilibrium dialysis (RED) is predominantly used\nfor the\ncharacterization of drug absorption, distribution, metabolism, and\nexcretion (ADME) properties in plasma and biological fluids. We describe\nherein improvements in the use of RED in conjunction with mass spectrometry\n(RED-MS) to enable robust binding affinity measurements of small molecules\nfor recombinant proteins and complexes from a single dialysis data\nset. The affinities calculated from RED-MS correlated well with measurements\nby both surface plasmon resonance (SPR) and affinity selection mass\nspectrometry (AS-MS). The method was particularly useful for quantifying\nthe binding of small molecules to large protein complexes that were\nnot amendable by common biophysical characterization techniques. Compound\npooling and integration with automated liquid handling increased assay\nthroughput and enabled the analysis of hundreds of measurements per\nweek. RED-MS offers a viable option for measuring compound binding\nin solution and may facilitate small molecule affinity optimization\ntoward difficult-to-drug protein complexes.