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A Whole New World of Native Mass Spectrometry

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If you could be granted one wish that would give you a lifetime of smooth-sailing protein purification, successful binding quantification and incredible insights for biotherapeutic analysis, what would it be?

A wise genie might encourage you to wish for the latest technology in native mass spectrometry (native MS), to directly characterize large protein complexes at unprecedented resolution.

Within the biopharmaceutical industry, large proteins are not only major targets of drugs, but they are also used as drugs themselves, in the form of monoclonal antibodies, antibody–drug conjugates and fusion proteins. Characterizing interactions between proteins and other molecules is crucial for understanding and modulating a protein’s function, guiding multiple stages of drug discovery and development. Due to technical limitations, proteins are typically assessed in isolation after being removed from their native environment. However, as proteins conduct their biological activities in complexes, this reductionist approach results in the loss of functionally important information.

Native MS has become a powerful technique over the past two decades and continues to evolve into a sophisticated tool for the analysis of large protein complexes. It is now beginning to attract significant attention from drug discovery companies wishing to exploit these capabilities. In this article, we share how modern native MS technology now offers deeper and more dynamic biological insights and explain why the corresponding platforms are a worthy choice for your “one wish”.

It’s not that simple: recognizing dynamics and diversity is key to protein analysis

Classical structural biology techniques, such as X-ray crystallography or cryogenic electron microscopy, provide information on binding and/or structural/functional aspects of a protein. By piecing together X-ray structures, researchers have gained a better understanding of the biology surrounding protein signaling. While these techniques are improving, the methods used to achieve high-resolution images obscure the structural heterogeneity in samples and fail to provide the whole picture.

Proteins regulate a diverse range of critical biological systems by forming both transient and stable interactions with other biomolecules. For example, a protein could engage in a brief interaction during a signaling cascade or form a key component of a multi-subunit molecular machine. These protein assemblies are complex and change their shape and binding capacity when exposed to different physical conditions, such as pH and temperature. To capture data reflective of a protein’s true function and state, protein complexes would ideally be characterized while preserved in their folded and bound state. As we will discuss later in this article, this is a particular challenge for the analysis of membrane proteins, which constitute the largest family of drug targets.

Interactions between proteins and small molecules are important aspects to consider when elucidating protein function, yet gathering information related to protein–ligand binding is challenging and complex. With traditional approaches to structural biology, important high definition details about protein–ligand binding are often lost, as:
  • Interactions can be transient and dynamic
  • Deciphering the exact chemical identity of lipids, metabolites and cofactors in contact with the protein is difficult
  • Polypeptides often undergo function altering covalent modifications (post-translational modification), creating additional complexity

Covalent modifications can significantly affect protein function. However, these modifications are usually minimized or removed during structural analyses. Although lipidomics and metabolomics methods can be used to identify small molecule protein binding partners, these methods often require previous knowledge of ligand chemistry and may involve large-scale screening or chemical extraction. Such interventions sever the link between non-covalent binding partners and the protein/complex, requiring subsequent confirmation of inferred ligands to be achieved by incubation with the original protein/complex and inferred ligand. Despite advances in these techniques, ligand chemical structures often remain unidentified.

Recently, native MS methods have been developed to overcome these challenges and limitations. Unraveling the chemical identity of a ligand binding partner of a protein/complex – i.e. identifying more than just its mass – is now an attainable goal. This is enabled by new MS instrumentation technology, which allows observation and isolation of the intact noncovalently bound ligand–protein complex, separation (dissociation) of the ligand from the protein/complex, followed by isolation and fragmentation of the liberated ligand for structural elucidation/identification all in one composite experiment. This has powerful implications for many branches of biology across academia and industry.

Pharma set to reap the rewards of advances in native mass spectrometry

In bottom-up proteomics, proteins are digested into peptides and typically separated via liquid chromatography before being delivered to the mass spectrometer. During sample preparation, the protein is denatured, leading to loss of non-covalent interactions. In contrast, in native MS, there is no protein digestion or denaturation, so the protein’s structural integrity and non-covalent associations are preserved, and intact protein complexes are delivered to the mass spectrometer. This allows direct observation of the entire complex, giving high resolution and accurate details on the composition and stoichiometry of the complex as well as bound endogenous ligands or synthetic compounds.

Studying proteins in their native state can provide deeper biological insights previously considered out of reach when working with traditional structural biology methods, such as the in-depth characterization of novel drug–protein interactions and ribosomal particles. As shown in Figure 1, modern native MS technology now provides a way to gather comprehensive data from large protein complexes.

Figure 1. Gathering comprehensive data from large protein complexes using native MS technology.

Native MS is increasingly utilized in academic research and in the biopharmaceutical industry at multiple stages of drug development. This is unsurprising, partly because high-resolution accurate-mass (HRAM) MS instruments, supported by software which eases data interpretation, have become more accessible and user-friendly. During biotherapeutic analysis, purification protocols must be assessed and optimized to ensure proteins are produced at a suitable yield and purity. Native MS is ideally suited to facilitate this assessment as it can provide information related to variants and impurities resulting from different cell culture conditions, gene expression, or other development conditions. For example, identifying lipids, truncated sequence variants, and protein homogeneity and stoichiometry can help guide purification and enable researchers to improve their processes. This information can also reveal how to preserve protein complexes in their functional state.

Quantitative data on ligand binding is extremely valuable during drug lead selection screening studies, where characterizing the binding affinities of ligands to proteins can guide candidate selection. In later stages of drug discovery, native MS can provide a host of valuable data for a range of biotherapeutics, including:
  • Therapeutic antibodies: e.g. sequence integrity, antigen-binding activity, glycan profile and characterization of other posttranslational modifications
  • Antibody–drug conjugates and bispecific antibodies: e.g. drug-to-antibody ratio, composition, and conjugation site information

Native MS opens doors for membrane protein research

Membrane proteins (such as G protein-coupled receptors (GPCRs), transporters and ion channels) fulfil a diverse range of biological functions and are highly valuable in the pharmaceutical industry, accounting for more than 50% of current drug targets. However, with their low expression levels and tendency to lose structural integrity outside of the lipid cellular membrane environment, the biophysical analysis of membrane proteins is particularly challenging. To support their structural integrity outside of the membrane, these proteins are formulated in lipophilic environments. Typically, this would occur in detergent micelles, but proteins can also be formulated in nanodiscs, amphipols, co-polymers or bicelles.

While the use of detergents and other stabilizers improve structural integrity of the protein, they do create further challenges across many biophysical techniques. In native MS, this challenge is overcome by vibrationally activating the proteomicelle complex while it is inside the mass spectrometer and releasing the protein from the detergent. With this approach, high-resolution mass measurements can be obtained to elucidate membrane protein stoichiometry and ligand binding interactions.       

Recently, native MS has provided unique insights by capturing and interrogating endogenous ligands within the environment of the intact protein assembly. In a study by Gault et al., using native MS technology developed by Oxford University spin-out OMass Therapeutics in close collaboration with scientists of Carol V. Robinsons’ laboratory at Oxford University, potential regulators of protein function were discovered and a host of molecular features of bound ligands were unraveled. In addition to protein homogeneity and confirmation, identification of the lipid class, chain length, extent of saturation, and peptide structure and therapeutic binding were gathered to ultimately characterize the functional lipidome/metabolome in contact with membrane porins and a mitochondrial translocator. This was achieved using a “nativeomics approach”, unifying native MS with “omics” analyses. Such interactions are often difficult to define using high resolution structural information alone, highlighting native MS as a powerful and complementary tool for membrane protein research.

The “nativeomics approach” has enabled an increased suite of applications for native MS; it can be used to define unknown electron density in high-resolution membrane protein maps and to discover the identity of key metabolites, ligands and cofactors associated with membrane protein transporters and receptors. When studying GPCRs, the ability to visualize and characterize protein activation enables researchers to concurrently:
  • Identify the protein recruited to transmit the signal
  • Quantify the strength of compound binding
  • Elucidate the consequences of ligand binding

While other methods provide information on binding or function, native MS combines both aspects in a high resolution “biophysical” tool.

No sign of native MS development slowing down

Looking ahead, native MS users have their sights set on a few future objectives, as more extensive analysis of co-purifying ligands would always be welcome to those seeking novel biological insights. Further development of binding and function reading of GPCRs is a key priority, which could be extended to other proteins of interest.

The capacity to analyze proteins/complexes directly from cellular environments by native MS would be the ultimate analytical goal for the field. As progress is made toward that ambitious goal, there is a suite of smaller technical and technological advancements that could be integrated with and utilized for native MS based analytics, such as more automated liquid handling capabilities and improved analytical software allowing streamlining of the analysis of complexes. Altogether, scientists will continue to address bottlenecks throughout the overall analytical protocol, from sample extraction and preparation to data processing and interpretation.

Granting a simple wish: to analyze large protein complexes with confidence

For a long time, obtaining information on many molecular features and interactions between proteins and endogenous ligands remained somewhat out of reach. However, significant efforts have been directed at advancing native MS techniques to overcome previous limitations of protein complex analysis. Modern native MS technology can benefit biopharmaceutical research and drug discovery by providing an enriched and high-resolution view of biological systems, which can be followed up with other complementary structural biology techniques, if needed.

High resolution data related to membrane protein–ligand interactions can now be readily obtained to determine the physiological pathways mediated by the respective interactions, providing a better chance of finding successful drug candidates. Native MS continues to advance, providing value in the context of protein purification, binding quantification and biotherapeutic characterization, and stands as a worthy choice should you encounter a genie in your analytical laboratory, or simply be open to an MS upgrade.

Article Authors:

Dr Krisztina Radi, Pharma & Biopharma Vertical Marketing Manager, Chromatography & Mass Spectrometry, Thermo Fisher Scientific

Dr Ali Jazayeri, Chief Scientific Officer, OMass Therapeutics