Chapter 11. Quantification of Protein–Ligand Interactions in Solution by Hydrogen/Deuterium Exchange (PLIMSTEX)

  1. Prof. Dr. Klaus T. Wanner and
  2. Dr. Georg Höfner
  1. Mei M. Zhu1,
  2. David Hambly2 and
  3. Michael L. Gross3

Published Online: 24 MAY 2007

DOI: 10.1002/9783527610907.ch11

Mass Spectrometry in Medicinal Chemistry: Applications in Drug Discovery

Mass Spectrometry in Medicinal Chemistry: Applications in Drug Discovery

How to Cite

Zhu, M. M., Hambly, D. and Gross, M. L. (2007) Quantification of Protein–Ligand Interactions in Solution by Hydrogen/Deuterium Exchange (PLIMSTEX), in Mass Spectrometry in Medicinal Chemistry: Applications in Drug Discovery (eds K. T. Wanner and G. Höfner), Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. doi: 10.1002/9783527610907.ch11

Editor Information

  1. Ludwig-Maximilians-University Munich, Department of Pharmacy, Center of Drug Research, Butenandtstr., 81377 Munich, Germany

Author Information

  1. 1

    Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, MA 02139, USA

  2. 2

    1201 Amgen Court West, Seattle, WA 98119, USA

  3. 3

    Center for Biomedical and Bioorganic Mass Spectrometry, Department of Chemistry, Washington University, Campus Box 1134, One Brookings Drive, St. Louis, MO 63130, USA

Publication History

  1. Published Online: 24 MAY 2007
  2. Published Print: 23 FEB 2007

Book Series:

  1. Methods and Principles in Medicinal Chemistry

Book Series Editors:

  1. Prof. Dr. Raimund Mannhold5,
  2. Prof. Dr. Hugo Kubinyi6 and
  3. Prof. Dr. Gerd Folkers7

Series Editor Information

  1. 5

    Molecular Drug Research Group, Heinrich-Heine-Universität, Universitätsstrasse 1, 40225 Düsseldorf, Germany

  2. 6

    Donnersbergerstrasse 9, 67256 Weisenheim am Sand, Germany

  3. 7

    Collegium Helveticum, STW/ETH Zurich, 8092 Zurich, Switzerland

ISBN Information

Print ISBN: 9783527314560

Online ISBN: 9783527610907

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Keywords:

  • quantification of protein–ligand interactions in solution by hydrogen/deuterium exchange (PLIMSTEX);
  • PLIMSTEX method;
  • applications of PLIMSTEX; determination of association constant (Ka);
  • stoichiometry (n) and protection (ΔDi); features of PLIMSTEX;
  • fast radical footprinting for protein–ligand interaction analysis;
  • fast photochemical oxidation of proteins

Summary

This chapter contains sections titled:

  • Introduction

  • The PLIMSTEX Method

    • A General Protocol of H/D Exchange and LC/MS Analysis for PLIMSTEX

    • Determination and Interpretation of the Titration Curves

  • Applications of PLIMSTEX

    • Determination of Association Constant (Ka), Stoichiometry (n), and Protection (ΔDi)

    • Ras-GDP Interacting with Mg2+: A 1:1 Protein:Metal Ion Interaction

      • Kinetic Study of Forward H/D Exchange Ras-GDP with Different [Mg2+]

      • PLIMSTEX Results for Ras-GDP Titrated with Mg2+

      • Interpretation of PLIMSTEX Results with H/D Exchange Kinetics

      • Application of PLIMSTEX to Relatively Weak Protein–Ligand Binding

      • Experimental Issues Regarding Using Metal Chelators

    • Apo-CaM Interacting with Ca2+: A 1:4 Protein:Metal Ion Interaction

      • PLIMSTEX Results for CaM and Intermediate Protein–Ligand Binding Species

      • PLIMSTEX in Biologically Relevant Media and High Ionic Strength

    • Apo-IFABP and Oleate: A Protein–Small Organic Molecule Interaction

    • Holo-CaM and Melittin: A Protein–Peptide Interaction

      • PLIMSTEX Curves Under Different Holo-CaM Concentrations

    • Self-association of Insulin: A Protein–Protein Interaction

      • Modified Version of PLIMSTEX for Insulin Self-association

  • Features of PLIMSTEX

    • Determines Ki, Stoichiometry, and Protection (ΔDi)

    • Requires Low Quantities of Protein

    • Relies Only on MS to Measure m/z And Not Solution Concentration

    • Works in Biologically Relevant Media at High Ionic Strength

    • Does Not Need Specially Labeled Protein or Ligand

    • Avoids Perturbation of the Binding Equilibrium

    • Has Potential for Peptide Resolution

    • Current Challenges and Future Directions

  • Fast Radical Footprinting for Protein–Ligand Interaction Analysis

    • Rationale for Hydroxyl Radicals as a Probe

    • Methods for Generating Hydroxyl Radicals

    • Fast Photochemical Oxidation of Proteins

    • Locating the Sites of Oxidation

    • Application of FPOP to Apomyoglobin

    • Advantages of FPOP

  • Potential Applications in Drug Discovery