Hydrogen/deuterium exchange mass spectrometry for investigating protein-ligand interactions

Amide hydrogen/deuterium exchange mass spectrometry is rapidly becoming a powerful method for high-resolution analyses of protein dynamics, structure, and function. Hydrogen/deuterium exchange approaches can provide information that greatly augments and refines information derived from high-resolution structural studies, and can provide detailed information on native protein structure when structural information is unavailable. Application of this method for rapid analyses of protein-ligand complexes could prove useful for studies of important disease-related protein complexes. The following review covers fundamentals of hydrogen/deuterium exchange and its applications to the study of protein-ligand complexes. In addition, hydrogen/deuterium exchange mass spectrometry studies on a protein-inhibitor complex are presented.     

An overview of hydrogen deuterium exchange mass spectrometry (HDX-MS) in drug discovery

Introduction: Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful methodology to study protein dynamics, protein folding, protein-protein interactions, and protein small molecule interactions. The development of novel methodologies and technical advancements in mass spectrometers has greatly expanded the accessibility and acceptance of this technique within both academia and industry.

Areas covered: This review examines the theoretical basis of how amide exchange occurs, how different mass spectrometer approaches can be used for HDX-MS experiments, as well as the use of HDX-MS in drug development, specifically focusing on how HDX-MS is used to characterize bio-therapeutics, and its use in examining protein-protein and protein small molecule interactions.

Expert opinion: HDX-MS has been widely accepted within the pharmaceutical industry for the characterization of bio-therapeutics as well as in the mapping of antibody drug epitopes. However, there is room for this technique to be more widely used in the drug discovery process. This is particularly true in the use of HDX-MS as a complement to other high-resolution structural approaches, as well as in the development of small molecule therapeutics that can target both active-site and allosteric binding sites.     

Molecular interactions between matrilysin and the matrix metalloproteinase inhibitor doxycycline investigated by deuterium exchange mass spectrometry

Matrix metalloproteinases (MMPs) play an essential role in normal and pathological extracellular matrix degradation. Deuterium exchange mass spectrometry (DXMS) was used to localize the binding regions of the broad-spectrum MMP inhibitor doxycycline on the active form of matrilysin (residues 95-267) and to assess alterations in structure induced by doxycycline binding. DXMS analyses of inhibitor-bound versus inhibitor-free forms of matrilysin reveal two primary sites of reduced hydrogen/deuterium exchange (residues 145-153; residues 193-204) that flank the structural zinc binding site. Equilibrium dialysis studies of doxycycline-matrilysin binding yielded a K(d) of 73 microM with a binding stoichiometry of 2.3 inhibitor molecules per protein, which compares well with DXMS results that show principal reduction in deuterium exchange at two sites. Lesser changes in deuterium exchange evident at the amino and carboxyl termini are attributed to inhibitor-induced structural fluctuations. Tryptophan fluorescence quenching experiments of matrilysin with potassium iodide suggest changes in conformation induced by doxycycline binding. In the presence of doxycycline, tryptophan quenching is reduced by approximately 17% relative to inhibitor-free matrilysin. Examination of the X-ray crystal structure of matrilysin shows that the doxycycline-binding site at residues 193 to 204 is positioned within the structural metal center of matrilysin, adjacent to the structural zinc atom and near both calcium atoms. These results suggest a mode of matrilysin inhibition by doxycycline that could involve interactions with the structural zinc atom and/or calcium atoms within the structural metal center of the protein.     

High-performance tandem mass spectrometry in metabolism studies

1. High-performance tandem mass spectrometry provides unit resolution in both selection of precursor ions and analysis of fragment ions, and extensive and reproducible fragmentation through collisional activation at high energy.

2. Metabolites can be analysed that occur as minor components in?h.p.l.c. peaks or other mixtures. Homogeneous isotopic species can be selected for unambiguous analysis of distributions of isotope labels. Fragmentation may be significantly enhanced to provide structural information. Overall, the signal to noise ratio is greatly improved and the spectrum is simplified.

3. These points are illustrated by isotope-labelling studies of the mechanisms of glutathione conjugation of the anti-tumour agent cyclophosphamide, the cytotoxic agent phosphoramide mustard and dimethylbilirubin, an analogue of bilirubin designed to be distinguishable from endogenous bilirubin. Analysis of isomeric mixed disulphides formed between glutathione and a peptide with an internal disulphide bond is discussed.

4. Reaction-induced decomposition is presented as an alternative to collisionally induced decomposition with more efficient energy transfer.     

Identification of two glucuronide metabolites of doxylamine via thermospray/mass spectrometry and thermospray/mass spectrometry/mass spectrometry

Analysis of a high-pressure liquid chromatography fraction containing two urinary glucuronide metabolites of doxylamine by thermospray mass spectrometry (TSP/MS) provided [MH]+ ions for each metabolite. TSP/MS/MS of the [MH]+ ions provided a fragment ion characteristic of these metabolites. The results demonstrate the utility of TSP/MS analysis for biologically derived glucuronide metabolites.     

High-performance tandem mass spectrometry in metabolism studies.

1. High-performance tandem mass spectrometry provides unit resolution in both selection of precursor ions and analysis of fragment ions, and extensive and reproducible fragmentation through collisional activation at high energy. 2. Metabolites can be analysed that occur as minor components in h.p.l.c. peaks or other mixtures. Homogeneous isotopic species can be selected for unambiguous analysis of distributions of isotope labels. Fragmentation may be significantly enhanced to provide structural information. Overall, the signal to noise ratio is greatly improved and the spectrum is simplified. 3. These points are illustrated by isotope-labelling studies of the mechanisms of glutathione conjugation of the anti-tumour agent cyclophosphamide, the cytotoxic agent phosphoramide mustard and dimethylbilirubin, an analogue of bilirubin designed to be distinguishable from endogenous bilirubin. Analysis of isomeric mixed disulphides formed between glutathione and a peptide with an internal disulphide bond is discussed. 4. Reaction-induced decomposition is presented as an alternative to collisionally induced decomposition with more efficient energy transfer.     

Identification of the degradation product of ezlopitant,a non-peptidic substance P antagonist receptor,by hydrogen deuterium exchange,electrospray ionization tandem mass spectrometry (ESI/MS/MS) and nuclear magnetic resonance (NMR) spectroscopy

The degradation product of ezlopitant was isolated from low specific activity material and identified by solution phase hydrogen/deuterium (H/D) exchange and electrospray ionization tandem mass spectrometry (ESI/MS/MS) to be an isopropyl peroxide analog of ezlopitant. The structure of the degradant was further confirmed by nuclear magnetic resonance (NMR) spectroscopy utilizing complete ¹H and ¹³C assignments. Studies were also performed to identify the factors responsible for the oxidative degradation of ezlopitant, which included salt form, storage conditions and salt formation solvent. Of all the variable studies over a 3 weeks period, only a change in the salt form prevented this oxidative degradation.     

Quantification of selected herbicides and chlorinated phenols in urine by using gas chromatography/mass spectrometry/mass spectrometry

We have developed a method for determining selected chlorinated phenols and phenoxy herbicides in urine. The process of preparing the samples includes acid hydrolysis, extraction with benzene, derivatization with diazoethane, and column chromatography cleanup. We quantify the more volatile compounds by using capillary column gas chromatography/positive chemical ionization/mass spectrometry/mass spectrometry. Less volatile compounds are quantified by using electron capture negative chemical ionization in a single stage mass spectrometry mode. Quality control samples are included in each analytical run, and the results demonstrate that the analytical system is in control. Positive values for the target analytes are determined on the basis of appropriate relative retention time, a signal-to-noise ratio greater than 3:1, and a calculated concentration greater than 1 ppb. We determine the chlorine isotope ratios for each compound to assess the presence or absence of interferences. This analytical method has been applied in a case-control study of 199 individuals to examine exposure to the 12 target analytes.     

The use of inductively coupled plasma mass spectrometry as a detector in drug metabolism studies

Background: The inherent properties of element selectivity combined with high sensitivity and structure independent response, make inductively coupled plasma mass spectrometry (ICP-MS) an interesting alternative detection technique in drug metabolism studies. Objective: The application of online separation with ICP-MS detection in drug metabolism studies is reviewed with focus on the merits and demerits of this detection technique. The prerequisite for inclusion in this review is that the study involves a separation technique hyphenated online to the ICP-MS detection. Result/conclusion: ICP-MS detection is found to be advantageous for analysis of all drug substances detectable by ICP-MS compared to radiochemical detection. Detectable drugs are limited to halogen-, sulfur-, metal- and metalloid-containing compounds. The drawback of interference from endogenous compounds on quantitative mass balance estimations of non-metal drugs is addressed. The potential of determining the stoichiometry in metallo-drug biomolecule interactions is pointed out by presenting examples of simultaneous monitoring of metals in metallo-drugs and intrinsic ICP-MS detectable elements in biomolecules. It is concluded that ICP-MS detection is an indispensable technique in drug metabolism studies of metallo-drugs, although the applicability for traditional drugs is limited.     

Syntheses of deuterated jasmonates for mass spectrometry and metabolism studies

Jasmonic acid and its metabolites play an essential role in the regulation of plant development and systemic defense responses. Isotopically labeled standards are required to quantify plant hormones for metabolism studies using mass spectrometry. A convenient method for the preparation of deuterated analogs of jasmonates is demonstrated. Modification of commercially available methyl jasmonate by base‐catalyzed proton/deuterium exchange or Wittig reaction introduces either two or three heavy atoms into a molecule. Copyright © 2005 John Wiley & Sons, Ltd.     

Stability of antibody drug conjugate formulations evaluated using solid-state hydrogen-deuterium exchange mass spectrometry

Antibody drug conjugates (ADCs) have been at the forefront in cancer therapy due to their target specificity. All the FDA approved ADCs are developed in lyophilized form to minimize instability associated with the linker that connects the cytotoxic drug and the antibody during shipping and storage. We present here solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) as a tool to analyze protein structure and matrix interactions for formulations of an ADC with and without commonly used excipients. We compared results of the ssHDX-MS with accelerated stability results using size-exclusion chromatography and determined that the former technique was able to successfully identify the destabilizing effects of mannitol and polysorbate 80. In comparison, Fourier-transform infrared spectroscopy results were inconclusive. The agreement between ssHDX-MS and stressed stability studies supports the potential of ssHDX-MS as a method of predicting relative stability of different formulations.     

Deuterated abscisic acid analogs for mass spectrometry and metabolism studies

Four analogs of abscisic acid (ABA) with deuterium atoms at non‐exchangeable positions have been synthesized to be used as standards for quantitation of the plant hormone ABA by mass spectrometry and also to be employed as substrates for metabolism studies. Deuterium atoms were introduced in the side chain of the molecule, at C‐4 and/or C‐5, by deuteride or hydride reduction of a propargylic alcohol, an intermediate in the synthesis. As well, deuterium labels were introduced at the C‐8′ position by conjugate addition of a Grignard reagent containing the label to a cyclohexadienone intermediate, affording specific isotopically labeled ABA molecules with one to five deuterium atoms. Copyright © 2002 John Wiley & Sons, Ltd.     

Turbulent Flow Chromatography TFC-tandem mass spectrometry supporting in vitro/vivo studies of NCEs in high throughput fashion

Turbulent Flow Chromatography (TFC) is a powerful approach for on-line extraction in bioanalytical studies. It improves sensitivity and reduces sample preparation time, two factors that are of primary importance in drug discovery.     

Uncatalyzed microwave deuterium exchange labeling of bleomycin A2

Bleomycin sulfate in D₂O was deuterated using microwave irradiation under catalyst free conditions. Following the removal of labile deuterium and purification, bleomycin A₂ with mass M + 1 to M + 7 was obtained. Successful selective uncatalyzed microwave deuterium exchange reactions on examples from the following classes of heterocycles are also described: imidazole, thiazole, indole, purine, and quinazoline. The described method was used as a test for non‐labile active protons. Copyright © 2004 John Wiley & Sons, Ltd.