Hydrogen Exchange Mass Spectrometry of Proteins

Fundamentals, Methods, and Applications

David D. Weis

• Publisher: Wiley
• Year: 2016
• Print ISBN: 9781118616499
• eBook ISBN: 9781118703694
• Edition: 1
• Topic: Naturwissenschaften
• Subtopic: Spektroskopie & Spektralanalyse

Hydrogen exchange mass spectrometry is widely recognized for its ability to probe the structure and dynamics of proteins. The application of this technique is becoming widespread due to its versatility for providing structural information about challenging biological macromolecules such as antibodies, flexible proteins and glycoproteins. Although the technique has been around for 25 years, this is the first definitive book devoted entirely to the topic.

Hydrogen Exchange Mass Spectrometry of Proteins: Fundamentals, Methods and Applications brings into one comprehensive volume the theory, instrumentation and applications of Hydrogen Exchange Mass Spectrometry (HX-MS) - a technique relevant to bioanalytical chemistry, protein science and pharmaceuticals. The book provides a solid foundation in the basics of the technique and data interpretation to inform readers of current research in the method, and provides illustrative examples of its use in bio- and pharmaceutical chemistry and biophysics

In-depth chapters on the fundamental theory of hydrogen exchange, and tutorial chapters on measurement and data analysis provide the essential background for those ready to adopt HX-MS.  Expert users may advance their current understanding through chapters on methods including membrane protein analysis, alternative proteases, millisecond hydrogen exchange, top-down mass spectrometry, histidine exchange and method validation. All readers can explore the diversity of HX-MS applications in areas such as ligand binding, membrane proteins, drug discovery, therapeutic protein formulation, biocomparability, and intrinsically disordered proteins.

1
Hydrogen Exchange: A Sensitive Analytical Window into Protein Conformation and Dynamics

Pernille Foged Jensen and Kasper D. Rand

Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark

1.1 Isotopic Exchange and the Study of Protein Conformation and Dynamics

Hydrogen exchange (HX) monitored by mass spectrometry (MS) is a powerful analytical method for investigation of protein conformation and dynamics. HX-MS monitors isotopic exchange of hydrogens in protein backbone amides and thus serves as a sensitive method for probing protein conformation and dynamics along the entire protein backbone (except for proline) (Figure 1.1).

Figure 1.1 Location of protein backbone amide hydrogens in Factor VIIa (PDB: 1DAN [1]). HX of the protein backbone amides (gray circles) occur spontaneously in a solution containing D₂O. The rates of exchange are highly dependent on hydrogen bonding and to a lesser extent solvent accessibility and thus report on protein conformation, dynamics, and intermolecular interactions.

Adapted from Ref. [2].

Historically, the monitoring of isotopic exchange in proteins has posed a technical challenge. Initial methodologies employed include measuring HX using the ultracentrifugation procedure of Kaj Ulrik Linderstrøm-Lang [3] and later on infrared [4] or UV spectroscopy [5]. These protocols were labor-intensive and only capable of measuring the summed (global) HX of labile sites in the protein. In the 1960s, Englander et al. [6] developed a method for monitoring isotopic exchange by liquid scintillation using the radioactive isotope, tritium (³H). Subsequently, the advent of one-dimensional nuclear magnetic resonance (NMR) spectroscopy enabled the measurement of HX at spectrally resolved amide linkages. The impact of the latter two approaches was, however, limited. HX studies of proteins underwent a significant resurgence following the development of high-resolution two-dimensional NMR methods. NMR is capable of resolving the majority of amide hydrogen signals of smaller proteins, thus increasing the number of amides through which to probe local conformational properties [7]. The combination of HX and multidimensional NMR spectroscopy remains an important tool, provided certain requirements are met such as the availability of sufficient soluble protein and backbone resonance assignments can be made (i.e., smaller proteins) [8]. Typical applications include studies of protein–ligand binding [9, 10], protein folding and unfolding [8, 11], and site-directed mutagenesis [12, 13].

A novel method for monitoring HX of proteins by mass spectrometry was introduced by Zhongqi Zhang and David L. Smith in 1993 [14] (Figure 1.2). The method was based in part on a protein fragmentation methodology developed by Rosa et al. [15] and improved by Englander et al. [16] capable of providing intermediate spatial resolution to isotopic exchange studies using radioactive tritium. In the original fragmentation methodology by Rosa et al., the acid-stable protease pepsin was used to digest tritium-labeled protein samples under HX quench conditions (pH 2.5 and 0°C), and tritium radioactivity was measured in peptides isolated on a cooled chromatographic LC system. Circumventing the use of tritium, Zhang et al. performed isotopic exchange of cytochrome c in excess deuterium and subjected samples of the exchange reaction to pepsin fragmentation at quench conditions. The resulting peptide mixture was loaded on a cooled chromatographic LC system interfaced with a mass spectrometer, and the time-resolved mass increase due to deuterium incorporation was monitored by mass spectrometry. Advantageously, deuterium situated in the labile groups of side chains or in the N-terminal amino group exchange far more rapidly than main chain amide hydrogens (see Section 1.2.1) at pH 2.5 and was thus back-exchanged with the protiated solvent of the LC system. Therefore, the deuterium content of each peptide at different time intervals was a direct measure of the HX rate of amide hydrogens in the peptide. The principles of this classic HX-MS experiment still form the basis for HX-MS type workflows used today.

Figure 1.2 The classical continuous labeling (bottom-up) HX-MS experiment. HX of an equilibrat...

Table of contents

• Cover
• Title Page
• Table of Contents
• List of Contributors
• Foreword
• Preface
• A Note about Nomenclature
• 1 Hydrogen Exchange
• 2 Hydrogen Exchange Mass Spectrometry Experimental Design
• 3 Data Processing in Bottom-Up Hydrogen Exchange Mass Spectrometry
• 4 Method Validation and Standards in Hydrogen Exchange Mass Spectrometry
• 5 Millisecond Hydrogen Exchange
• 6 Proteases for Hydrogen Exchange Mass Spectrometry
• 7 Extracting Information from Hydrogen Exchange Mass Spectrometry Data
• 8 Gas-Phase Fragmentation of Peptides to Increase the Spatial Resolution of the Hydrogen Exchange Mass Spectrometry Experiment
• 9 Top-Down Hydrogen Exchange Mass Spectrometry
• 10 Histidine Hydrogen Exchange for Analysis of Protein Folding, Structure, and Function
• 11 Hydrogen Exchange Mass Spectrometry for the Analysis of Ligand Binding and Protein Aggregation
• 12 Application of Differential Hydrogen Exchange Mass Spectrometry in Small Molecule Drug Discovery
• 13 The Role of Hydrogen Exchange Mass Spectrometry in Assessing the Consistency and Comparability of the Higher-Order Structure of Protein Biopharmaceuticals
• 14 Utility of Hydrogen Exchange Mass Spectrometry in Epitope Mapping
• 15 Hydrogen Exchange Mass Spectrometry for Proteins Adsorbed to Solid Surfaces, in Frozen Solutions, and in Amorphous Solids
• 16 Hydrogen Exchange Mass Spectrometry of Membrane Proteins
• 17 Analysis of Disordered Proteins by Hydrogen Exchange Mass Spectrometry
• 18 Hydrogen Exchange Mass Spectrometry as an Emerging Analytical Tool for Stabilization and Formulation Development of Therapeutic Monoclonal Antibodies
• Index
• End User License Agreement