Throughput and efficiency of a mass spectrometry-based screening assay for protein—Ligand binding detection

Abstract

An H/D exchange-and MALDI mass spectrometry-based screening assay was applied to search for novel ligands that bind to cyclophilin A, a potential therapeutic and diagnostic target in lung cancer. The assay is based on stability of unpurified j;!roteins from rates of H/D exchange (SUPREX), which exploits the H/D exchange properties of amide protons to measure the increase in a protein's thermodynamic stability upon ligand binding in solution.The current study evaluates the throughput and efficiency with which 880 potential ligands from the Prestwick Chemical Library (Illkirch, France) could be screened for binding to cyclophilin A. Screening was performed at a rate of 3 min/ligand using a conventional MALDI mass spectrometer. False positive and false negative rates, based on a set of control data, were as low as 0% and 9%, respectively. Based on the 880-member library screening, a false positive rate of 0% was observed when a two-tier selection strategy was implemented. Although novel ligands for cyclophilin A were not discovered, cyclosporin A, a known ligand to CypA and a blind control in the library, was identified as a hit. We also describe a new strategy to eliminate some of the complications related to back exchange that can arise in screening applications of SUPREX. (J

Determination of thermodynamic and kinetic properties of biomolecules by mass spectrometry

“…In a typical SUPREX setting, a series of protein or proteinligand complex samples is incubated for a certain time in a deuterated buffer containing increasing amount of a chemical denaturant, for example, guanidinium hydrochloride. Hence, the extent of hydrogen exchange is analyzed as a function of denaturant concentration and yields the free energy of folding DG f and the ligandinduced stabilization of protein structure DDG f , although with some limitations [48,54,59]. Recently, a very similar method in which labile deuterium labeling is substituted with stable oxidative labeling of methionine residues has been proposed -SPROX (stability of proteins from rates of oxidation) [48,[60][61][62].…”

Painting proteins with covalent labels: What’s in the picture?

“…The results of a recent HTS experiment conducted in our laboratory demonstrated the speed and efficiency with which SUPREX can be used to screen large numbers of ligands for binding to a target protein [69]. This work used a single-point SUPREX protocol [70] in which binding events were detected by measuring the target protein's mass change after H/D exchange in a single SUPREX buffer containing a specific chemical denaturant concentration.…”

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

“…In cases where the error associated with the protein mass measurement in the single-point SUPREX protocol is not small compared to the number of globally protected amide protons in the protein, the efficiency of the assay is low (i.e., it can be difficult to discriminate between hits and non-hits). For example, the efficiency of the CypA screen in our recent work2 was significantly reduced in comparison with the screening efficiency found for S-protein in our original proof-of-concept study 1. Since CypA and S-protein have similar numbers of globally protected amide proteins (as judged by the amplitudes of their SUPREX curves), the reduced efficiency was attributed to the errors associated with the MALDI mass measurements on CypA (i.e., ± ~6 Da) being larger than those on S-protein (i.e., ± 1.5 Da).…”

“…We recently reported a MALDI-MS-based technique for the high-throughput detection of protein-ligand binding interactions 1,2. The technique, hereafter referred to as single-point SUPREX, is an abbreviated version of SUPREX (stability of unpurified proteins from rates of H/D exchange), which is an H/D exchange- and mass spectrometry-based approach for evaluating the free energy difference between the folded and unfolded forms of a protein (i.e., the folding free energy) 3,4.…”

“…In a recent application of single-point SUPREX, we screened an 880-member compound library for binding to cyclophilin A (CypA)2 and identified an important limitation of the single-point SUPREX protocol. The efficiency of the protocol was found to be highly dependent on the precision of the mass measurements and on the number of globally protected amide protons in the protein.…”