Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

Molecular Insights into the Thermal Stability of mAbs with Variable-Temperature Ion-Mobility Mass Spectrometry.

The aggregation of protein-based therapeutics such as monoclonal antibodies (mAbs) can affect the efficacy of the treatment and can even induce effects that are adverse to the patient. Protein engineering is used to shift the mAb away from an aggregation-prone state by increasing the thermodynamic stability of the native fold, which might in turn alter conformational flexibility. We have probed the thermal stability of three types of intact IgG molecules and two Fc-hinge fragments by using variable-temperature ion-mobility mass spectrometry (VT-IM-MS). We observed changes in the conformations of isolated proteins as a function of temperature (300-550 K). The observed differences in thermal stability between IgG subclasses can be rationalized in terms of changes to higher-order structural organization mitigated by the hinge region. VT-IM-MS provides insights into mAbs structural thermodynamics and is presented as a promising tool for thermal-stability studies for proteins of therapeutic interest.

Application of the Exactive Plus EMR for automated protein-ligand screening by non-covalent mass spectrometry.

RATIONALE: Non-covalent mass spectrometry (MS) offers considerable potential for protein-ligand screening in drug discovery programmes. However, there are some limitations with the time-of-flight (TOF) instrumentation typically employed that restrict the application of non-covalent MS in industrial laboratories. METHODS: An Exactive Plus EMR mass spectrometer was investigated for its ability to characterise non-covalent protein-small molecule interactions. Nano-electrospray ionisation (nanoESI) infusion was achieved with a TriVersa NanoMate. The transport multipole and ion lens voltages, dissociation energies and pressure in the Orbitrap™ were optimised. Native MS was performed, with ligand titrations to judge retention of protein-ligand interactions, serial dilutions of native proteins as an indication of sensitivity, and a heterogeneous protein analysed for spectral resolution. RESULTS: Interactions between native proteins and ligands are preserved during analysis on the Exactive Plus EMR, with the binding affinities determined in good agreement with expected values. High spectral resolution allows baseline separation of adduct ions, which should improve the accuracy and limit of detection for measuring ligand interactions. Data are also presented showing baseline resolution of glycoforms of a highly glycosylated protein, allowing binding of a fragment molecule to be detected. CONCLUSIONS: The high sensitivity and spectral resolution achievable with the Orbitrap technology confer significant advantages over TOF mass spectrometers, and offer a solution to current limitations regarding throughput, data analysis and sample requirements. A further benefit of improved spectral resolution is the possibility of using heterogeneous protein samples such as glycoproteins for fragment screening. This would significantly expand the scope of applicability of non-covalent MS in the pharmaceutical and other industries.

Dynamics of intact immunoglobulin†G explored by drift-tube ion-mobility mass spectrometry and molecular modeling.

Collision cross-sections (CCS) of immunoglobulins G1 and G4 have been determined using linear drift-tube ion-mobility mass spectrometry. Intact antibodies and Fc-hinge fragments present with a larger range of CCS than proteins of comparable size. This is rationalized with MD simulations, which indicate significant in vacuo dynamics between linked folded domains. The IgG4 subclass presents over a wider CCS range than the IgG1 subclass.

Identification of differential protein binding affinities in an atropisomeric pharmaceutical compound by noncovalent mass spectrometry, equilibrium dialysis, and nuclear magnetic resonance.

Atropisomerism of pharmaceutical compounds is a challenging area for drug discovery programs (Angew. Chem., Int. Ed. 2009, 48, 6398-6401). Strategies for dealing with these compounds include raising the energy barrier to atropisomerization in order to develop the drug as a single isomer (Tetrahedron 2004, 60, 4337-4347) or reducing the barrier to rotation and developing a mixture of rapidly interconverting isomers (Chirality 1996, 8, 364-371). Commonly, however, the atropisomers will be differentiated in terms of their affinity for a given protein target, and it is therefore important to rapidly identify the most active component prior to further compound development. We present equilibrium dialysis and saturation transfer difference NMR (STD-NMR) as techniques for assessing relative affinities of an atropisomeric mixture against antiapoptotic protein targets Bcl-2 and Bcl-xL. These techniques require no prior separation of the mixture of compounds and are therefore rapid and simple approaches. We also explore the use of noncovalent mass spectrometry for determining KD values of individual atropisomers separated from the equilibrium mixture and compare the results to solution-phase measurements. Results from equilibrium dialysis, STD-NMR, and noncovalent mass spectrometry are all in excellent agreement and provide complementary information on differential binding, amplification of the strongest binders, and KD values.

Mass spectrometry based tools to investigate protein-ligand interactions for drug discovery.

The initial stages of drug discovery are increasingly reliant on development and improvement of analytical methods to investigate protein-protein and protein-ligand interactions. For over 20 years, mass spectrometry (MS) has been recognized as providing a fast, sensitive and high-throughput methodology for analysis of weak non-covalent complexes. Careful control of electrospray ionization conditions has enabled investigation of the structure, stability and interactions of proteins and peptides in a solvent free environment. This critical review covers the use of mass spectrometry for kinetic, dynamic and structural studies of proteins and protein complexes. We discuss how conjunction of mass spectrometry with related techniques and methodologies such as ion mobility, hydrogen-deuterium exchange (HDX), protein footprinting or chemical cross-linking can provide us with structural information useful for drug development. Along with other biophysical techniques, such as NMR or X-ray crystallography, mass spectrometry provides a powerful toolbox for investigation of biological problems of medical relevance (204 references).

Atropisomeric small molecule Bcl-2 ligands: determination of bioactive conformation.

The separation of atropisomeric conformers of 1,2,3,4-tetrahydroisoquinoline amide Bcl-2 ligands allowed the identification of the bioactive conformer which was subsequently confirmed by X-ray crystallography.

Hybrid Mass Spectrometry Approaches to Determine How L-Histidine Feedback Regulates the Enzyzme MtATP-Phosphoribosyltransferase.

MtATP-phosphoribosyltransferase (MtATP-PRT) is an enzyme catalyzing the first step of the biosynthesis of L-histidine in Mycobacterium tuberculosis, and proposed to be regulated via an allosteric mechanism. Native mass spectrometry (MS) reveals MtATP-PRT to exist as a hexamer. Conformational changes induced by L-histidine binding and the influence of buffer pH are determined with ion mobility MS, hydrogen deuterium exchange (HDX) MS, and analytical ultracentrifugation. The experimental collision cross-section (DTCCSHe) decreases from 76.6 to 73.5 nm2 upon ligand binding at pH 6.8, which correlates to the decrease in CCS calculated from crystal structures. No such changes in conformation were found at pH 9.0. Further detail on the regions that exhibit conformational change on L-histidine binding is obtained with HDX-MS experiments. On incubation with L-histidine, rapid changes are observed within domain III, and around the active site at longer times, indicating an allosteric effect.

Development of a therapeutic monoclonal antibody that targets secreted fatty acid-binding protein aP2 to treat type 2 diabetes.

The lipid chaperone aP2/FABP4 has been implicated in the pathology of many immunometabolic diseases, including diabetes in humans, but aP2 has not yet been targeted for therapeutic applications. aP2 is not only an intracellular protein but also an active adipokine that contributes to hyperglycemia by promoting hepatic gluconeogenesis and interfering with peripheral insulin action. Serum aP2 levels are markedly elevated in mouse and human obesity and strongly correlate with metabolic complications. These observations raise the possibility of a new strategy to treat metabolic disease by targeting serum aP2 with a monoclonal antibody (mAb) to aP2. We evaluated mAbs to aP2 and identified one, CA33, that lowered fasting blood glucose, improved systemic glucose metabolism, increased systemic insulin sensitivity, and reduced fat mass and liver steatosis in obese mouse models. We examined the structure of the aP2-CA33 complex and resolved the target epitope by crystallographic studies in comparison to another mAb that lacked efficacy in vivo. In hyperinsulinemic-euglycemic clamp studies, we found that the antidiabetic effect of CA33 was predominantly linked to the regulation of hepatic glucose output and peripheral glucose utilization. The antibody had no effect in aP2-deficient mice, demonstrating its target specificity. We conclude that an aP2 mAb-mediated therapeutic constitutes a feasible approach for the treatment of diabetes.

Automated protein-ligand interaction screening by mass spectrometry.

Identifying protein-ligand binding interactions is a key step during early-stage drug discovery. Existing screening techniques are often associated with drawbacks such as low throughput, high sample consumption, and dynamic range limitations. The increasing use of fragment-based drug discovery (FBDD) demands that these techniques also detect very weak interactions (mM K(D) values). This paper presents the development and validation of a fully automated screen by mass spectrometry, capable of detecting fragment binding into the millimolar K(D) range. Low sample consumption, high throughput, and wide dynamic range make this a highly attractive, orthogonal approach. The method was applied to screen 157 compounds in 6 h against the anti-apoptotic protein target Bcl-x(L). Mass spectrometry results were validated using STD-NMR, HSQC-NMR, and ITC experiments. Agreement between techniques suggests that mass spectrometry offers a powerful, complementary approach for screening.