Hydrogen/deuterium exchange- and protease digestion-based screening assay for protein-ligand binding detection

A protease digestion strategy was incorporated into single-point stability of unpurified proteins from rates of H/D exchange (SUPREX), which is a hydrogen/deuterium (H/D) exchange- and mass spectrometry-based assay for the detection of protein-ligand binding. Single-point SUPREX is an abbreviated form of SUPREX in which protein-ligand binding interactions are detected by measuring the increase in a protein's thermodynamic stability upon ligand binding. The new protease digestion protocol provides a noteworthy increase in the efficiency of single-point SUPREX because peptide masses can be determined with greater precision than intact protein masses in the matrix-assisted laser desorption ionization (MALDI) readout of single-point SUPREX. The protocol was evaluated in test screens on two model protein systems, including cyclophilin A (CypA) and the minor allele variant of human alanine:glyoxylate aminotransferase (AGTmi). The test screening results obtained on both proteins revealed that the peptide readout of the single-point SUPREX-protease digestion protocol was more efficient than the intact protein readout of the original single-point SUPREX protocol at discriminating hits and nonhits. In addition to this improvement in screening efficiency, the protease digestion strategy described here is expected to significantly increase the generality of the single-point SUPREX assay.