Hydrogen/Deuterium Exchange- and Protease Digestion-Based Screening Assay for Protein−Ligand Binding Detection

Abstract

A protease digestion strategy was incorporated into single-point SUPREX (stability of unpurified proteins from rates of H/D exchange), which is an 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 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 non-hits. 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.

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

“…Beyond providing structural and kinetic insight, pulse labeling has also been useful for characterization of protein ligand binding properties. By varying ligand concentration, it is possible to extract binding affinities , and assess the effects of numerous ligands on protein stability. , These approaches have paved the way to high throughput studies to identify inhibitors against therapeutic protein targets …”

Peptide-Level Interactions between Proteins and Small-Molecule Drug Candidates by Two Hydrogen−Deuterium Exchange MS-Based Methods: The Example of Apolipoprotein E3

“…Reasons to choose SUPREX are that it can provide information at the peptide or regional level and it has high sensitivity. High sequence coverage is not necessarily required because a single peptide with a well-defined SUPREX curve is sufficient to represent the global or subglobal stability change induced by ligand binding . Representative peptides in domains of interest should be subglobally protected, insensitive to local fluctuations, and not solvent-accessible …”

“…High sequence coverage is not necessarily required because a single peptide with a well-defined SUPREX curve is sufficient to represent the global or subglobal stability change induced by ligand binding. 23 Representative peptides in domains of interest should be subglobally protected, insensitive to local fluctuations, and not solvent-accessible. 22 Our modification of SUPREX is motivated to characterize denaturation of various regions of the C-terminal domain where binding of the ligand occurs, to gain more information about ligand binding.…”

“…The binding constant can then be calculated from the ΔΔG f between bound and unbound states. 19–21 Thermodynamics of domains 22 or of large peptides 23 can also be probed with SUPREX coupled with enzyme digestion.…”

“…Multiple C 1/2 SUPREX determinations at varied exchange times are used to calculate an m value (i.e., dependence of folding free energy change on denaturant) and Δ G f (i.e, the folding free energy in the absence of denaturant). The binding constant can then be calculated from the ΔΔ G f between bound and unbound states. − Thermodynamics of domains or of large peptides can also be probed with SUPREX coupled with enzyme digestion.…”