Journal of the American Society for Mass Spectrometry

, Volume 19, Issue 9, pp 1303–1311 | Cite as

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

  • Erin D. Hopper
  • Petra L. Roulhac
  • Michael J. Campa
  • Edward F. Patz
  • Michael C. FitzgeraldEmail author


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 proteins 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.


False Negative Rate Cyclophilin Library Compound MALDI Mass Spectrum Denaturant Concentration 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Papac, D. I.; Shahrokh, Z. Mass Spectrometry Innovations in Drug Discovery and Development. Pharm. Res. 2001, 18, 131–145.CrossRefGoogle Scholar
  2. 2.
    Shin, Y. G.; van Breemen, R. B. Analysis and Screening of Combinatorial Libraries Using Mass Spectrometry. Biopharm. Drug Dispos. 2001, 22, 353–372.CrossRefGoogle Scholar
  3. 3.
    Sussmuth, R. D.; Jung, G. Impact of Mass Spectrometry on Combinatorial Chemistry. J. Chromatogr. B. 1999, 725, 49–65.CrossRefGoogle Scholar
  4. 4.
    Ghaemmaghami, S.; Fitzgerald, M. C.; Oas, T. G. A. Quantitative, High-Throughput Screen for Protein Stability. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8296–8301.CrossRefGoogle Scholar
  5. 5.
    Ma, L. Y.; Fitzgerald, M. C. A New H/D Exchange- and Mass Spectrometry-Based Method for Thermodynamic Analysis of Protein-DNA Interactions. Chem. Biol. 2003, 10, 1205–1213.CrossRefGoogle Scholar
  6. 6.
    Powell, K. D.; Fitzgerald, M. C. Accuracy and Precision of a New H/D Exchange- and Mass Spectrometry-Based Technique for Measuring the Thermodynamic Properties of Protein-Peptide Complexes. Biochemistry 2003, 42, 4962–4970.CrossRefGoogle Scholar
  7. 7.
    Powell, K. D.; Ghaemmaghami, S.; Wang, M. Z.; Ma, L.; Oas, T. G.; Fitzgerald, M. C. A General Mass Spectrometry-Based Assay for the Quantitation of Protein-ligand Binding Interactions in Solution. J. Am. Chem. Soc. 2002, 124, 10256–10257.CrossRefGoogle Scholar
  8. 8.
    Powell, K. D.; Wang, M. Z.; Silinski, P.; Ma, L.; Wales, T. E.; Dai, S. Y.; Warner, A. H.; Yang, X.; Fitzgerald, M. C. The Accuracy and Precision of a New H/D Exchange- and Mass Spectrometry-Based Technique for Measuring the Thermodynamic Stability of Proteins. Anal. Chim. Acta. 2003, 496, 225–232.CrossRefGoogle Scholar
  9. 9.
    Tang, L.; Hopper, E. D.; Tong, Y.; Sadowsky, J. D.; Peterson, K. J.; Gellman, S. H.; Fitzgerald, M. C. H/D Exchange- and Mass Spectrometry-Based Strategy for the Thermodynamic Analysis of Protein-ligand Binding. Anal. Chem. 2007, 79, 5869–5877.CrossRefGoogle Scholar
  10. 10.
    Powell, K. D.; Fitzgerald, M. C. High-Throughput Screening Assay for the Tunable Selection of Protein Ligands. J. Combinat. Chem. 2004, 6, 262–269.CrossRefGoogle Scholar
  11. 11.
    Campa, J.; Wang, M. Z.; Howard, B.; Fitzgerald, M. C.; Patz, E. F. Protein Expression Profiling Identifies Macrophage Migration Inhibitory Factor and CyclophilinA as Potential Molecular Targets in Non-small Cell Lung Cancer. Cancer Res. 2003, 63, 1652–1656.Google Scholar
  12. 12.
    Howard, B. A.; Furumai, R.; Campa, M. J.; Rabbani, Z. N.; Vujaskovik, Z.; Wang, X. F.; Patz, E. F. Stable RNA Interference-Mediated Suppression of Cyclophilin A Diminishes Non-Small-Cell Lung Tumor Growth in Vivo. Cancer Res. 2005, 65, 8853–8860.CrossRefGoogle Scholar
  13. 13.
    Handschumacher, R. E.; Harding, M. W.; Rice, J.; Drugge, R. J. Cyclophilin: A Specific Cytosolic Binding-Protein for Cyclosporin A. Science 1984, 226, 544–547.CrossRefGoogle Scholar
  14. 14.
    Harding, M. W.; Handschumacher, R. E. Cyclophilin, a Primary Molecular Target for Cyclosporine—Structural and Functional Implications. Transplantation 1988, 46, S29-S35.CrossRefGoogle Scholar
  15. 15.
    Holzman, T. F.; Egan, D. A.; Edalji, R.; Simmer, R. L.; Helfrich, R.; Taylor, A.; Burres, N. S. Preliminary Characterization of a Cloned Neutral Isoelectric form of the Human Peptidyl Prolyl Isomerase Cyclophilin. J. Biol. Chem. 1991, 266, 2474–2479.Google Scholar
  16. 16.
    Liu, J.; Albers, M. W.; Chen, C. M.; Schreiber, S. L.; Walsh, C. T. Cloning, Expression, and Purification of Human Cyclophilin in Escherichia coli and Assessment of the Catalytic Role of Cysteines by Site-Directed Mutagenesis. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 2304–2308.CrossRefGoogle Scholar
  17. 17.
    Wang, M. Z.; Shetty, J. T.; Howard, B. A.; Campa, M. J.; Patz, E. F.; Fitzgerald, M. C. Thermodynamic Analysis of Cyclosporin A Binding to Cyclophilin A in a Lung Tumor Tissue Lysate. Anal. Chem. 2004, 76, 4343–4348.CrossRefGoogle Scholar
  18. 18.
    Taylor, D. M. Radioiodinated Cyclosporin A: Preparation and Biodistribution. Int. J. Appl. Radiat. Isot. 1980, 31, 192–193.CrossRefGoogle Scholar
  19. 19.
    Bai, Y. W.; Milne, J. S.; Mayne, L.; Englander, S. W. Primary Structure Effects on Peptide Group Hydrogen-Exchange. Prot. Struct. Funct. Genet. 1993, 17, 75–86.CrossRefGoogle Scholar
  20. 20.
    Zhang, Y. Z. Structural Biology and Molecular Biophysics; Ph.D. Thesis, University of Pennsylvania, 1995.Google Scholar
  21. 21.
    Schellman, J. A. Macromolecular Binding. Biopolymers 1975, 14, 999–1018.CrossRefGoogle Scholar
  22. 22.
    Glasoe, P. K.; Long, F. A. Use of Glass Electrodes to Measure Acidities in Deuterium Oxide. J. Phys. Chem. 1960, 64, 188–190.CrossRefGoogle Scholar
  23. 23.
    Nozaki, Y. The Preparation of Guanidine Hydrochloride. Methods Enzymol. 1972, 26, 43–50.CrossRefGoogle Scholar
  24. 24.
    Zhang, J. H.; Chung, T. D. Y.; Oldenburg, K. R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J. Biomol. Screen. 1999, 4, 67–73.CrossRefGoogle Scholar
  25. 25.
    Powell, K. D.; Fitzgerald, M. C. Measurements of Protein Stability by H/D Exchange and Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Using Picomoles of Material. Anal. Chem. 2001, 73, 3300–3304.CrossRefGoogle Scholar
  26. 26.
    Hann, M. M.; Oprea, T. I. Pursuing the Leadlikeness Concept in Pharmaceutical Research. Curr. Opin. Chem. Biol. 2004, 8, 255–263.CrossRefGoogle Scholar
  27. 27.
    Oprea, T. I. Current Trends in Lead Discovery: Are We Looking for the Appropriate Properties?. J. Comput. Aided Mol. Des. 2002, 16, 325–334.CrossRefGoogle Scholar
  28. 28.
    Zhang, J.-H.; Wu, X.; Sills, M. A. Probing the Primary Screening Efficiency by Multiple Replicate Testing: A Quantitative Analysis of Hit Confirmation and False Screening Results of a Biochemical Assay. J. Biomol. Screen. 2005, 10, 695–704.CrossRefGoogle Scholar
  29. 29.
    Tang, L.; Roulhac, P. L.; Fitzgerald, M. C. H/D Exchange and Mass Spectrometry-Based Method for Biophysical Analysis of Multidomain Proteins at the Domain Level. Anal. Chem. 2007, 79, 8728–8739.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2008

Authors and Affiliations

  • Erin D. Hopper
    • 1
  • Petra L. Roulhac
    • 1
  • Michael J. Campa
    • 2
  • Edward F. Patz
    • 2
  • Michael C. Fitzgerald
    • 1
    Email author
  1. 1.Department of ChemistryDuke UniversityDurhamUSA
  2. 2.Department of RadiologyDuke University Medical CenterDurhamUSA

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