Proteomics reaches critical mass

Can mass spectrometry do for complex protein research what it did for small molecule analysis? Dr Jonathan Hopper thinks a trick is missed unless we draw on this staple to expose the workings of molecular machines involved in disease

Proteins are the functional workhorses of the cells, driving all major biological processes through cascades of interactions and communication networks, or by self-assembling into huge molecular machines. Unfortunately, the malfunction of these molecular machines result in disease. This makes them the focal point for pharmaceutical and medical researchers around the world.

Scientists are now harnessing proteins that are produced naturally in response to infection, such as antibodies, to attack disease

Of course, unpicking the secrets of protein structures is not an easy task – they are complex and change in response to different conditions. To further compound these difficulties, as well as being the targets for therapeutic drugs, proteins are now being increasingly used as the therapeutic themselves. Scientists are now harnessing proteins that are produced naturally in response to infection, such as antibodies, to attack disease. Although these biologics possess remarkable abilities, their protein structure and modifications must be controlled and regulated with stringent precision to produce consistent results. This is where native mass spectrometry (native MS) provides powerful insights.

Mass spectrometry has been around as an analytical tool for decades, providing unparalleled mass accuracy for determining the composition of small molecules, which has made it one of the cornerstone techniques of analytical science. However, native MS now allows scientists to apply this same level of accuracy to comparatively massive and complex protein molecules.

It also allows folded proteins and their binding partners to remain intact as they transit through the vacuum of the spectrometer – which allows the interactions of proteins to be captured. By preserving large protein structures, scientists can understand how these molecules communicate and assemble into the molecular machines that drive the key functions in cells. This not only enables the direct measurement of assembly and composition, but also the way important proteins interact with one another – vitally important for pharmaceutical and biotech industries.

A dynamic problem

Some protein structures are highly dynamic, consisting of flexible components that are necessary for them to accomplish their roles within the cell. These dynamics can pose two main problems. Firstly, isolating these proteins away from their native cellular environments is often accompanied by a loss in structural stability. Secondly, proteins that don’t adopt a well-ordered structure are often intractable to some of the most widely used techniques for determining structure. For example, X-ray crystallography requires the molecules to form well-ordered crystal lattices. However, native MS does not require proteins to adopt rigid conformations and is able to sample a range of dynamic states. Moreover, since the technique only requires relatively small amounts of material, proteins that are unstable and difficult to isolate in large quantities are amenable to the approach.

In the membrane

Another challenge in research is the difficulty of analysing membrane proteins. For pharmaceutical and medical research, membrane proteins constitute an extremely valuable class of drug targets. They reside in the ‘greasy’ walls (membranes) of cells and are responsible for a wide range of sensory responses. Of all the available drugs currently on the market, approximately 60% target this class of proteins. But their native, hydrophobic environment means that membrane proteins are not soluble in water – a major restrictive barrier for most scientific methods.

Scientists largely negate these problems using additives that serve to mimic the hydrophobic environment of the cell membrane; the most widely used being detergent molecules. Detergent molecules possess both a hydrophobic component, which interacts with protein, and a soluble component that keeps the protein solubilised.

It was discovered that the detergent could be used to ‘protect’ complex membrane protein assemblies as they are introduced into the spectrometer

The breakthrough for native MS was made when protocols were introduced that allow the technology to be used with membrane proteins decorated in detergent molecules. It was discovered that the detergent could be used to ‘protect’ complex membrane protein assemblies as they are introduced into the spectrometer. Once safely inside the mass spectrometer, detergent is removed in a controlled way to yield an intact membrane protein complex in the gas phase for interrogation. Following this, research began with pharmaceutical companies on extremely difficult targets for which little or no structural information was available. The collaborations led to an understanding that this technique can provide vital information for many different aspects of medical research, ranging from early stage pre-clinical work on drug targets, to harnessing the power and speed of MS for ‘quality control’ applications around antibody purification.

Maturing MS

To accommodate this need, my team at OMass (a start-up spun out of Oxford University) now provide the enabling platform and consultancy necessary to drive the technology into pharmaceutical research. Some of the case studies, which have already emerged using native MS have already demonstrated the great potential of the technology in pharmaceutical research.

Native MS has now matured into a technique that can help to inform pharmaceutical researchers on many different levels. The increasing complexity of drug targets, coupled with the need to provide ever more data for regulatory purposes, is fuelling the need for new technologies and platforms, which address these challenges. Just as traditional MS has become the cornerstone in the analysis of small molecules, so too will this level of information become accessible to protein scientists and molecular biologists.

Now is a very exciting time to be involved in native MS. As work with research scientists in biotechnology companies and in drug discovery continues, new insights on challenging targets that were inconceivable only a few years ago, are emerging.

[box type="shadow" align="aligncenter" ]   The proof is in the pilot studies…

• The potential of drug screening studies using native MS has been demonstrated during early research where it was possible to assess the ability of small molecules to stabilise the four protein subunits in transthyretin (involved in amyloid disease)¹. This research helped initiate a structure-based drug design program for small molecules that led eventually to the discovery of Tafamidis, in the Jeffery Kelly Laboratory. Tafamidis was later acquired by Pfizer.

• Native MS also contributed to the development of bifunctional crosslinking ligands for linking protein subunits in C-reactive protein², transthyretin3 and serum amyloid P⁴. These targets are important in heart disease, amyloid disease and Alzheimer’s disease respectively. This was in collaboration with Mark Pepys and colleagues at Pentraxin Therapeutics, a spin out company from University College London.

• One of the most challenging and important drug targets, G-Protein Coupled Receptors (GPCRs) can now be studied in great detail using native MS. In our latest publication, our researchers have shown how the platform allows interactions between receptors and potential drug molecules to be captured (Science Advances in press). Unexpectedly, it was also uncovered how these drugs influence signalling pathways within the cell to achieve their therapeutic effects. New unpublished data demonstrates the ability to capture GPCR interactions with nanobodies and how lipids play an intimate role in the function of these receptors; key information for in-vitro pharmaceutical research.

• As well as uncovering the beneficial mechanisms of drugs, native MS is also able to provide information regarding unwanted side-effects. For example, whilst studying the interactions of the dugs lopinavir and ritonavir, intended for HIV therapy, researchers in the Robinson laboratory found they blocked a critical enzyme pathway linked to Progeria-like symptoms, which cause premature aging⁵. [/box]

Author: Dr Jonathan Hopper is CEO at OMass

References: 1: McCammon, M. G. et al. Screening transthyretin amyloid fibril inhibitors: characterization of novel multiprotein, multiligand complexes by mass spectrometry. Structure 10, 851-863 (2002). 2: Pepys, M. B. et al. Targeting C-reactive protein for the treatment of cardiovascular disease. Nature 440, 1217-1221, doi:10.1038/nature04672 (2006). 3: Mangione, P. P. et al. Bifunctional crosslinking ligands for transthyretin. Open biology 5, 150105, doi:10.1098/rsob.150105 (2015). 4: Kolstoe, S. E. et al. Molecular dissection of Alzheimer's disease neuropathology by depletion of serum amyloid P component. Proceedings of the National Academy of Sciences of the United States of America 106, 7619-7623, doi:10.1073/pnas.0902640106 (2009). 5: Mehmood, S., Marcoux, J. & Gault, J. Mass spectrometry captures off-target drug binding and provides mechanistic insights into the human metalloprotease ZMPSTE24. 8, 1152-1158, doi:10.1038/nchem.2591 (2016).