H/D Exchange- and Mass Spectrometry-Based Strategy for the Thermodynamic Analysis of Protein−Ligand Binding

Tang, Liangjie; Hopper, Erin D.; Tong, Yan; Sadowsky, Jack; Peterson, Kimberly J.; Gellman, Samuel H.; Fitzgerald, Michael C.

doi:10.1021/ac0700777

Cited by 45 publications

(53 citation statements)

References 41 publications

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“…45 The dissociation constants for rapamycin binding to both wild-type and F99L FKBP-12, derived using the capillary technique, are compared Table II. Thermodynamic Parameters for the Denaturation of BSA, FKBP-12 wt and FKBP-12 F99L 40,43,44 The microplate-based fluorescence technique gave a value of 12.8 nM, and the capillary technique gave 7.4 nM, both consistent with the existing literature data.…”

Section: Resultsmentioning

confidence: 99%

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

et al. 2010

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Protein stability and ligand-binding affinity measurements are widely required for the formulation of biopharmaceutical proteins, protein engineering and drug screening within life science research. Current techniques either consume too much of often precious biological or compound materials, in large sample volumes, or alternatively require chemical labeling with fluorescent tags to achieve measurements at submicrolitre volumes with less sample. Here we present a quantitative and accurate method for the determination of protein stability and the affinity for small molecules, at only 1.5-20 nL optical sample volumes without the need for fluorescent labeling, and that takes advantage of the intrinsic tryptophan fluorescence of most proteins. Coupled to appropriate microfluidic sample preparation methods, the sample requirements could thus be reduced 85,000-fold to just 10 8 molecules. The stability of wild-type FKBP-12 and a destabilizing binding-pocket mutant are studied in the presence and absence of rapamycin, to demonstrate the potential of the technique to both drug screening and protein engineering. The results show that 75% of the interaction energy between FKBP-12 and rapamycin originates from residue Phe99 in the binding site.

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Section: Resultsmentioning

confidence: 99%

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

et al. 2010

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“…Thus, larger proteins that have SUPREX curves with large amplitudes may still be amenable to single-point SUPREX assays. The amplitude of a SUPREX curve tends to be slightly larger when short exchange times are used [9]; therefore, for larger proteins, the Z=-factor could potentially be improved slightly by adjusting the exchange time.…”

Section: Efficiencymentioning

confidence: 99%

“…We recently reported on a mass spectrometry-based assay for protein-ligand binding detection that utilizes an abbreviated version of stability of unpurified j;!roteins by rates of H/D exchange (SUPREX), which is an H/D exchange-and mass spectrometry-based technique capable of detecting and quantifying protein-ligand binding interactions [4][5][6][7][8][9]. In the abbreviated version of SUPREX (referred to hereafter as single-point SUPREX), binding events are detected by measuring the target protein's mass change after H/D exchange in a deuterated buffer containing a specific concentration of a chemical denaturant [10].…”

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confidence: 99%

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

Hopper

Roulhac

Campa

et al. 2008

J. Am. Soc. Mass Spectrom.

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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

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“…In cases where the global unfolding/refolding reaction of a protein is well modeled by a two-state transition (i.e., partially folded intermediate states of the protein are not populated), SUPREX provides reasonably accurate ⌬G f and m-values for the transition [24,27,31,32]. Such SUPREX-derived ⌬G f and m-values are especially useful for the quantitation of protein-ligand binding affinities.…”

Section: The Informationmentioning

confidence: 99%

“…Such SUPREX-derived ⌬G f and m-values are especially useful for the quantitation of protein-ligand binding affinities. SUPREX measurements of a protein's ⌬G f and m-value made in the presence and in the absence of a ligand can be used to calculate a ⌬⌬G f value between the protein and protein-ligand complex (i.e., the binding free energy) [27,31,32,62,63].…”

Section: The Informationmentioning

confidence: 99%

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

Fitzgerald

West

2009

J. Am. Soc. Mass Spectrom.

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Knowledge about the structural and biophysical properties of proteins when they are free in solution and/or in complexes with other molecules is essential for understanding the biological processes that proteins regulate. Such knowledge is also important to drug discovery efforts, particularly those focused on the development of therapeutic agents with protein targets. In the last decade a variety of different covalent labeling techniques have been used in combination with mass spectrometry to probe the solution-phase structures and biophysical properties of proteins and protein-ligand complexes. Highlighted here are five different mass spectrometry-based covalent labeling strategies including: continuous hydrogen/deuterium (H/D) exchange labeling, hydroxyl radical-mediated footprinting, SUPREX (stability of unpurified proteins from rates of H/D exchange), PLIMSTEX (protein-ligand interaction by mass spectrometry, titration, and H/D exchange), and SPROX (stability of proteins from rates of oxidation). The basic experimental protocols used in each of the above-cited methods are summarized along with the kind of biophysical information they generate. Also discussed are the relative strengths and weaknesses of the different methods for probing the wide range of conformational states that proteins and proteinligand complexes can adopt when they are in solution. (J Am Soc Mass Spectrom 2009, 20, 1193-1206) © 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry P roteins fold into elaborate three-dimensional structures and, under native solution conditions, they spend a large fraction of time in highly compact structures. However, the solution-phase structures of proteins are not static. Proteins sample a wide range of conformational states in solution. The conformational changes that proteins undergo in solution can be as dramatic as a global unfolding event in which all higher-order structure is lost or as subtle as a breathing motion in which a specific element of secondary structure is partially unfolded in a more local unfolding event (see Figure 1). Knowledge about the structures, kinetics, and thermodynamics involved in the conformational changes that proteins undergo in solution and as they interact with ligands is crucial for understanding the fundamental biological processes in which proteins participate. It is also important to drug discovery efforts, particularly those focused on the development of therapeutic agents with protein targets.Mass spectrometry (MS) has become an increasingly useful analytical tool for acquiring biophysical information about the conformational properties of proteins in solution. A common strategy that has emerged for acquiring such information is to use matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI) MS to read out the results of solutionphase reactions that introduce covalent modifications into proteins. Two general types of MS-based covalent labeling strategies have proven useful for probing the biophysical p...

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H/D Exchange- and Mass Spectrometry-Based Strategy for the Thermodynamic Analysis of Protein−Ligand Binding

Cited by 45 publications

References 41 publications

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

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

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

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