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Native Mass Spectrometry: A Glimpse Into the Machinations of Biology

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Image courtesy of the Heck laboratory, Utrecht University, The Netherlands
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Native mass spectrometry (native MS) is defined as the process whereby large biomolecules and complexes thereof can be transferred from a three-dimensional, functional existence in a condensed liquid phase to the gas phase via the process of electrospray ionization mass spectrometry (ESI-MS). The experimental conditions where this is possible are so mild that non-covalent interactions, the native state and the associated biological action and functionality of the molecule/complex are largely preserved. Historically, native MS is around 27 years old; with the first pioneering studies on non-covalent complexes appearing in the literature in the early 1990s.1,2 This initial work was closely followed by investigators such as Joseph A. Loo (UCLA) and Carol V. Robinson (Oxford University). Together the research of these scientists planted the initial seeds of native MS as a futuristic method in structural biology. Their early publications focused on protein–protein interactions3; protein–ligand complexes;3,4 mass spectra of the molecular chaperone GroEL;5 nanomachines such as ribosomes;6 and even intact viruses.7

Led by innovators such as Albert J.R. Heck, native MS continues to grow, develop and improve; particularly in terms of mass resolution and sensitivity.8,9 Heck reflects on his inspiration to pursue MS as a technique for determining protein structure: “Dudley Williams gave a lecture where the interaction of vancomycin with peptides had been measured by NMR. Although successful, the approach was not easy; and I thought, perhaps such questions could be addressed by MS?” The concept captured Heck’s attention and directed his early interests in native biological MS; and later, into pushing the mass limits of the methodology. Shown in the figure below is the remarkable evolution of native MS from early beginnings to the present day.

The evolution of native mass spectrometry. From the early beginnings of small proteins; through to present day supramolecular complexes of intact virus particles and bacteriophages. Image courtesy of the Heck laboratory, Utrecht University, The Netherlands.

Much ado about native MS

Native MS is an incredibly useful approach to gain insights into the behavior of proteins under physiological conditions. By harnessing an analytical technique such as MS and applying the technology to biological situations, it has become much easier to grasp what is occurring at the molecular level in a cell. Although a controlled and somewhat contrived approach, native MS nevertheless highly-reflects the natural in vivo state of a molecule or complex. Therefore, significantly more meaningful information is generated on structure-function relationships that can then be used in combination with molecular modeling and, for example, in the design of drugs etc.

Fundamentally, native MS is the precision technique that can provide biological context. One of the forefathers of native MS, Joseph A. Loo, tells us: “Think about it…the function of most biological macromolecules is dictated by the interactions they form. Recent human “interactome” research suggests that the average number of interactors for each protein is around ten. And that’s part of the normal function of every protein encoded by the genome.” He believes that native MS has the potential to provide a means to directly study such interactions that are the drivers of protein function and is convinced that with more researchers adopting native MS, new exciting applications will be reported on a routine basis.

The success of native MS is defined by two key factors

To reliably conduct a native MS experiment, there are key criteria that must be addressed to ensure success. The first criterion is the nature of the solution containing the native complex of interest. To maintain structural integrity and prevent degradation, most protein complexes are stored under physiologically-compatible conditions. Essentially, this means in buffers that are non-volatile and contain salts, surfactants and other stabilizers that are usually not compatible with the process of electrospray ionization. For most proteomic applications, maintaining complex integrity and non-covalent interactions is not required. As such, denaturing conditions are employed whereby proteins are unfolded via the addition of agents that destroy any pre-existing three-dimensional structure of the macromolecular complexes.

Thus, an essential requirement for observing intact native protein complexes by MS is to exchange the usual physiological buffer to a volatile ammonium-based semi-buffer that not only maintains integrity of the interactions; but is also compatible with the desolvation process required during ESI to transfer molecules from liquid to gas phase. Achieving these fundamentals is not always straightforward, as pointed out by Loo: “Proper sample preparation still remains one of the biggest hurdles for many laboratories.” Furthermore, he goes on to say that: “It’s often sample dependent, so how one type of sample is treated may not be applicable for another type.” Although he acknowledges that significant progress has been made in the past 10 years and vendors are now offering viable solutions to address sample preparation, “It’s still a significant issue for the field.”

Secondly, as the protein complexes are in a natural folded state, the number of available sites for acquiring a charge is reduced, and subsequently the number of visible charge states in the mass spectrum also decreases. The downside is that these large supramolecular complexes have very high mass-to-charge (m/z) ratios that are generally beyond the mass range of standard mass spectrometers. To further native MS, it was important that instruments were developed with advanced physical features and capabilities. Led by Standing, Robinson, and Heck the quadrupole time-of-flight (QTOF) mass spectrometer was the instrument of choice for analyzing large macromolecular assemblies.5-7, 10, 11

In 2012, a collaborative effort between Alexander A. Makarov and the Heck laboratory reported developments in native MS on an orbitrap mass spectrometer.8 Modifications across the entire instrument were accommodated, thereby boosting ion performance in the high m/z range. In addition, the important issues of high resolution, e.g., baseline-resolved peaks from molecular mass increases of <0.1 % of the total mass, and sensitivity were also addressed. To this aspect, Loo comments: “Today, mass spectrometers have been designed and optimized specifically for native MS, and many researchers have a good understanding of how to operate the instruments to maximize performance. It’s still not routine, but it’s definitely getting there!” 

The unparalleled diversity of native MS applications

The question perhaps is not when native MS can be used; but rather, when can’t it be used? Today, the applications are broad-reaching and diverse. In proteomics, native MS is well-suited to observe and analyze non-covalent biomolecular complexes. Experimental systems range from the study of: intact proteins including assessing protein isoforms within a mixture and defining post-translational modifications; intact protein/subunit folding dynamics; intact protein conformations and conformational changes; protein complexes and stoichiometry (particularly of wild-type versus mutant); protein complex assembly states; biologically-active complexes and determining protein binding affinity constants. Furthermore, other areas of key interest include observing and understanding conformational changes that occur in proteins (or DNA/RNA) when a complex is formed with a small molecule (such as a drug or metabolite) and/or nucleic acids.

Small molecule-protein interactions have always been of interest to academia and the pharmaceutical industry alike. Loo was one of the first people to comprehend the power of native MS applications in this sector. During a sojourn from academia, he realized that: “Native MS could be exploited to further drug discovery efforts because small molecule drugs usually target proteins. Thus, native MS could be used to characterize protein-drug complexes and aid development of more effective drug compounds.”

The subsequent adoption of native MS into the biotechnology and biopharmaceutical sectors heralded the transition of this important technique from an academic research tool to a more routine application. In addition to the on-going interest in small molecule discovery and development, this sector has a vested interest in protein therapeutics. Thus, it was imperative that a robust approach was implemented to characterize antibody-based immunoconjugates, biosimilars, and antibody-drug conjugates. As Heck says: “Although native MS is 20 to 25 years old; it has only relatively recently become a part of the toolbox for Pharma and acceptance within the industry is rapidly expanding”. Quality control assessment of impurities and/or aggregates in such products is critical to obtaining approval by the governing authorities.

For example, analysis of a sample under non-denaturing conditions not only confirms the molecular mass of the product, but also provides invaluable information on homogeneity, conformation (folded versus unfolded), and oligomerization state; all of which are key factors in generating a quality product. In addition, analysis under native conditions may also reveal the existence of natural or unexpected ligands that may have co-purified with the protein sample. According to Loo: “Native MS still has a ways to go to impact Pharma in a really meaningful way, but with more therapeutic proteins in the pipeline, native MS of antibody drugs is a growing application.” He further says: “Instrumentation still needs to improve, overall sensitivity needs to improve, and sampling throughput needs to increase.” Loo’s words are echoed by Heck who adds that: “Data analysis is central, and bioinformatics must also improve to keep abreast of pharmaceutical applications.” These factors will undoubtedly be key to ensuring native MS has a permanent and central position in biological pharmaceutical production pipelines of the future.

The perspectives for native MS are astonishing: dream BIG!

Native MS is highly-complementary to almost all the tools that are currently used in structural biology. From relatively small quantities of starting material, this technique can rapidly provide fundamental information to a structural biologist. Furthermore, clever integration of native MS with data generated from other sources, e.g., cross-linking MS, hydrogen-deuterium exchange MS, surface labeling and more recently, cryo-EM, provides a wealth of information that leads to the generation of structures that truly emulate the in vivo situation.

Heck’s prediction of the future is that: “There will be a reduction in sample preparation, and it will be possible to analyze protein structures in situ – direct from cell to MS.” In addition, he believes that: “There is no limitation on how big you can go,” for example, mass cytometry is a method that already uses ESI on whole cells. Rather he says that the question should be: “What sort of useful biological information can we get? And with what precision?”

With respect to the scientific community, Loo says: “I’m optimistic that how much native MS grows depends only on the creative imagination of everyone in the field.” Although still in the realm of today’s science fiction, he suggests that: “When combined with methods such as top-down MS, native MS can be THE tool for determining high-resolution protein structures.” He goes on to say: “More than 27 years have elapsed since a protein complex was first analyzed by ESI-MS; and look at all the progress protein MS has made since those days. One can (and should) still dream BIG.”


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