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Native mass spectrometry is a technique that aims to keep biological structures as close to their natural state as possible during analysis. As such, it is widely used to analyze complex protein assemblies. Preserving the structure of the protein assemblies is unlike conventional reversed-phase LC/MS techniques, since the biomolecule’s folded state and any subsequent noncovalent interactions are kept intact. Therefore, protein interactions due to noncovalent electrostatic, hydrophobic, van der Waals, or hydrogen bonding effects can be examined using native MS. This allows for a more accurate picture to be developed, enabling researchers to understand the vital role of these interactions in biological functions.
One of the latest innovations in native MS is that it can now be used to analyze membrane proteins. Membrane proteins are large biological molecules located in or on the periphery of the lipid bilayer of cells. These proteins are responsible for transmitting signals across the membrane and transporting substances in and out of the cell. Approximately half of all small drug molecules bind to membrane proteins on the cell’s surface, making their study so important. Because membrane proteins are expressed within a highly hydrophobic surface, they first need to be extracted.
A powerful extraction technique from Oxford Mass Technologies (OMass, Oxfordshire, U.K.) allows intact membrane protein assemblies to be introduced to the mass spectrometer. The platform is the first to analyze membrane proteins using native MS techniques, as well as important membrane protein drug targets such as G protein-coupled receptors (GPCRs) and receptor kinases.
Membrane protein analysis
Native MS studies liquid phase interactions, but MS is an analysis technique that requires samples to be in the gas phase. To provide information on the solution structure of proteins, electrospray ionization (ESI) avoids thermally activating these complexes, allowing the noncovalent interactions to be preserved. The MS measurement is only a few milliseconds and therefore enables the protein structures to be detected in the gas phase before any significant structural changes can occur. This approach gives a glimpse of the sample’s structure(s) as they would be naturally, and allows them to be characterized accordingly.
By carefully selecting the correct detergents and finely tuning the gas-phase manipulation, OMass was able to successfully sample membrane proteins directly in native-like environments. Direct detection of proteins and protein–ligand complexes were achieved using a mass spectrometer purposefully developed to meet the unique demands and specifications from OMass—the Thermo Scientific Q Exactive UHMR Hybrid-Quadrupole Orbitrap mass spectrometer. The system was previously used for analysis of ions with mass-to-charge ratios up to 80,000 m/z. Thus, it is possible that even larger protein complexes could soon be analyzed under native conditions.
Unique structural information can be obtained from this technique. The charge state distribution of the protein structures can be observed, because the folded protein architecture gives rise to low and narrow peak distributions. Ion mobility can also provide further information regarding the orientationally averaged cross-section. This ion mobility method is able to capture any major conformational changes and dynamics within the structure and adds further detail to traditionally two-dimensional MS datasets. This, in combination with other labeling approaches, such as hydrogen-deuterium exchange (HDX) and chemical cross-linking, can capture residue specific changes (such as ligand binding) and allows the changes in dynamics to be seen and the binding sites to be mapped.
Native MS and drug discovery
Membrane proteins are extremely difficult to analyze using traditional biophysical techniques; however, it is essential to study them for drug discovery. Cutting-edge native MS technologies can aid the drug discovery process from early to late stage and across a range of programs:
- Protein purification: often in drug discovery research, producing enough membrane protein targets can be lengthy and labor-intensive. Native MS can provide a direct measure of the homogeneity and stoichiometry of the protein with high resolution, while also reporting on any lipids or impurities present from preparation steps.
- Binding quantification: When characterizing a drug candidate, it is often useful to test the binding affinities of the ligands to proteins or protein complexes. Native MS provides quantitative information on this for a single protein, or for a large protein assembly, by monitoring discrete changes in mass with relative signal intensities.
- Biotherapeutic analysis: In the late stages of drug discovery, native MS is extremely useful for the characterization of therapeutic antibodies. The native MS platform developed by OMass can quickly assess the integrity of a sequence, the antigen binding profile, and the glycan profile. These intricate analyses can be performed in a single measurement, and with only picomoles of material.
Native MS and protein–lipid interactions
Interactions between proteins and lipids are more complex and more important than ever. Rather than simply anchoring proteins in the membrane, lipids can interact in a targeted way with proteins in order to directly change their structure and function. Native MS is therefore ideally suited to study the effects of protein–lipid binding by using its ability to examine copurified lipids and introduce defined endogenous lipids, in addition to resolving all binding populations simultaneously. It allows interactions to be studied in terms of affinity or conferred structural stability.
Alternative techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) are more challenging for complex equilibria. Developing native MS as a complementary approach to existing methods means that extremely challenging systems can be studied. For example, GPCRs and ion channels are two such protein classes that are highly sought-after as drug targets for a range of diseases. Systems like these can possess complex interaction networks that consist of cofactors of other biomolecules, in addition to the candidate drug. Complex characterization requires techniques that are able to sample multiple coexisting states simultaneously. In addition, the analysis must be performed with sufficient resolution so that the populations of interest can be observed. This is achievable using OMass native MS in conjunction with the Q Exactive UHMR system.
Native MS and biotherapeutics
Pharmaceutical research increasingly focuses on proteins; by harnessing their superior specificity and efficacy, they can be used as therapeutic agents. However, compared to small-molecule drugs, they are much more complex and present several challenges for analysts because of their potential for heterogeneity from variations in production, processing, or storage. There are demands to studying proteins in a preclinical R&D environment, as well as controlling the quality of the resulting drug products. By performing direct mass measurements of folded antibodies from aqueous solutions, native MS provides rapid access to key characteristics, such as sequence fidelity. Also, any truncations, impurities, or causes of heterogeneity are immediately apparent.
For antibodies, various glycoforms of the protein can be resolved, and relative abundances can be quantified. With native MS, a single spectrum provides this information in minutes—much faster than orthogonal methods of analysis. Antibodies can be analyzed in the presence of the target receptor, which can confirm activity under the same conditions. OMass native MS also employs HDX as part of preclinical R&D to identify the region or regions of a receptor involved in binding to a specific antibody. The technique eliminates the need for costly and laborious structural methods.
Conclusion
The native MS technique described here offers advances in several areas of drug discovery. From membrane protein targets, to protein–lipid interactions, to biologics, the mass resolution achievable enables confident characterizations of complex protein interactions.
Dr. Kelly Broster is pharma & biopharma manager, Thermo Fisher Scientific, 1 Boundary Park, Hemel Hempstead, HP2 7GE, U.K.; e-mail:[email protected]; www.thermofisher.com/nativems