Native Mass Spectrometry Facilitates Hit Validation in DNA-Encoded Library Technology.

DNA-encoded Library (DEL) Technology has become a workhorse of drug development, is widely employed in an industrial and academic setting, and an increasing number of drugs developed by DEL technology have entered clinical stage development. While up to billions of compounds can be screened simultaneously in affinity-based selections, the validation and characterization of individual hits discovered from DEL selections is a substantial bottleneck since it can be cumbersome and time-consuming. Here, we describe the use of native mass spectrometry for the speedy hit validation of On-DNA compounds. Through the preservation of noncovalent interactions in the gas phase, complexes of proteins and their respective On-DNA ligands can be analyzed without further need of labeling or immobilization. By utilizing the workflow described in this work, we were able to reliably rank affinities of various purified On-DNA binders or demonstrate binding of On-DNA compounds from unpurified mixtures, mitigating the need for tedious purification steps. Additionally, the methodology described here can offer valuable insight on which moiety of a binding molecule contributes the most to binding, facilitating subsequent medicinal chemistry efforts for lead expansion.