EN / DA
Disease and treatment

How life’s building blocks got their freedom of movement

The myriad processes that take place in our body’s cells are the basis of life as we know it. Since cells are filled with fluid, British chemist Carol Robinson created a lot of attention by setting out to investigate life’s processes in a vacuum. Despite massive resistance, she persevered. Now she is receiving the Novozymes Prize for founding a new subfield of mass spectrometry to investigate the shape of proteins and how they interact. Today, this technique is used to identify brand-new targets for drugs.

Some of the most important proteins in our bodies are on the surface of all cells. The membrane proteins control the transport in and out of cells and are key to how cells communicate. This makes membrane proteins hugely important drug targets. However, since the proteins are situated inside hydrophobic membranes, researchers have had great difficulty in figuring out how the proteins assemble and interact with other molecules.

Carol Robinson is receiving the 2019 Novozymes Prize for her scientific breakthroughs in using mass spectrometry – especially her pioneering work on using mass spectrometry for analysing protein complexes.

“The membrane proteins are the really critical complexes and are incredibly hard to study, because of this whole phase issue, where they sit in the oily phase. What we did was to coat them in detergent – effectively in soap bubbles – and then we release them into a mass spectrometer. Miraculously, they stay intact in a folded stage, so we can examine their 3D structure but also how they bind to other proteins or lipids,” explains Carol Robinson, Professor, Department of Chemistry, University of Oxford.

An unusual path to success

Mass spectrometry was originally developed to determine the mass of small molecules. In a mass spectrometer, the molecules are ionized and deflected in a magnetic field during their flight. Heavy molecules are hardly affected in their flight, whereas lighter ones are deflected more. Measuring how far the ions fly can determine how much they weigh. The molecules are sorted based on their mass-to-charge ratio.

“I am still hugely fascinated with seeing a beam of ions fly through a mass spectrometer and what you can learn from the spectra. A mass spectrometry spectrum is a bit like a sudoku puzzle. You start to get all possibilities and then you can solve the problem and you feel tremendously satisfied.”

Carol Robinson ending up as the first woman chemistry professor at Oxford was not a given, and her journey has been quite unusual. After leaving school at 16, she joined Pfizer as a technician working in the mass spectrometry laboratory. One day, one of Carol’s colleagues recognized that she had very special potential.

“They encouraged me to do seven years of part-time study. It was the long haul, and then I was delighted to be accepted at the University of Cambridge to do a PhD. Traditionally, mass spectra were just used to determine mass. In my PhD project, I was looking at how we could also use the mass spectrometer to determine the sequence of small fragments of a protein.”

Molecular elephants with wings

Since proteins are built from specific sequences of the 21 amino acids – 21 different building blocks with different masses – the researchers managed to identify the protein sequences by looking at the distance between the peaks in the spectra. Carol Robinson’s career was on track, but then something happened.

“I did something that was considered rather unusual. I took a career break for eight years, which was bit unconventional in those days. I really enjoyed the time at home with my three children and then went back to Oxford.”

At 35 years old, she returned to science through a junior position working on mass spectrometry in Chris Dobson’s group at the University of Oxford. While she was away, some revolutionary things had happened in mass spectrometry.

“I was encouraged to enter the new field of proteomics that emerged at that time. But we were not just looking at peptides and sequencing the amino acids along small chains. We were now looking at whole proteins, which are huge in terms of mass spectrometry.”

Instead of being just a few hundred mass units (daltons), the proteins are between 20,000 and 30,000 daltons.

“The challenge was how to make these huge molecules fly in the mass spectrometer. John Fenn, one of my science heroes who received part of the Nobel Prize for discovering electrospray mass spectrometry, tried putting it like this: ‘Well, I gave molecular elephants wings’.“

Spray-painting the proteins

Although Fenn got the very large protein molecules to fly, Carol Robinson wanted more – much more. She wanted to use the mass spectrometer to determine the very shape of the protein. She started to work on structural proteomics very early after the electrospray ionization technology became available. This enabled intact proteins to be introduced from solution to vacuum.

She got a crazy idea: if she could spray-paint the molecules, she could separate them based on how much paint they were covered by. The ultimate goal was to monitor protein-folding reactions by using mass spectrometry.

“Spray-painting an unfolded protein would take a lot of paint. But if it is folded into a very compact structure and then painted, I would not use so much paint, and that is exactly what we do. We spray it with deuterium, which weighs more than hydrogen. If an area of a protein is unfolded, hydrogen atoms are exposed and exchanged with deuterium. But if it is tightly folded, it won’t, so it weighs less.”

Carol Robinson developed a hydrogen-deuterium exchange method that could detect contact surfaces on proteins and applied it to study the folding of proteins. With this technique, protein structures could be preserved in vacuum, and this laid the basis for studying the structure of large proteins as well as protein–protein interactions using mass spectrometry.

A damning commentary

Carol Robinson used this technique to determine the folding state of a protein. However, her scientific colleagues thought she was crazy. At the molecular level, life as we know it occurs in water. A mass spectrometer has a vacuum.

“So it was really quite a damning commentary, and it was in a very respected journal: Proceedings of the National Academy of Sciences in the United States, so actually it was very hard to publish, because I would always have this cited when I submitted my research papers. People would say, ‘Well, haven’t you read this, it is a crazy idea.’ I would say: ‘Yes, I know, but I really believe it’.”

In her quest to prove that mass spectrometers could be used to detect even large intact proteins, Carol Robinson had to break the existing mould of what was possible. This required developing a tandem mass spectrometer with improved ion transmission and a higher mass-to-charge range than before.

“At that time, the spectrometers typically took particles up to about 4000 mass-to-charge ratio. We thought we would be a bit revolutionary and go up to 32,000. That is a huge jump, and I remember people sort of cautioning me against that. They said just go to 8000. I said: ‘No, 32,000 would allow us to do so much,’ and I got one made and I bought it.”

The new mass spectrometers proved Carol Robinson’s point that protein complexes retain their structure in a vacuum; they also enabled researchers to detect intact protein complexes with a molecular mass above 500,000 daltons and to study subunit organization.

“Our experiments clearly established that protein complexes retain their subunit stoichiometry in the mass spectrometer. Today, these instruments are available from several mass spectrometry manufacturers and are widely applied in the pharmaceutical industry to investigate intact protein biopharmaceuticals, such as antibodies and membrane receptors, but at that time it was a small revolution.”

A big breakthrough

Rather than just trying to show that they could do clever things with a mass spectrometer, Carol Robinson chose to take things to a new level.

“We thought maybe we can now answer some really key questions: for example, how the proteins come together in complexes. If we disrupted them, maybe they would fall apart in pairs, in threes or any other combination. And we could then tell from these interactions how they were assembled.”

This new method enabled Carol Robinson and her colleagues to study how large protein complexes assemble and how proteins interact with co-factors and other proteins. In a pioneering work, she determined the conformation of GroEL, an important protein chaperone in the bacterium Escherichia coli that is highly conserved in humans.

“Chaperones protect other proteins while they are folding, creating a sort of protective environment. Our technique enabled us to examine its amazing structure. It has 14 copies of the same protein that form these two rings, and inside is the folding protein, and we started to look at how this protected environment would change folding.”

Ever since then, Carol Robinson’s group has studied even more important and complex structures such as binding interactions within an antibody–antigen complex, and this method is now used routinely for characterizing antibodies in the pharmaceutical industry.

“This has enabled more rapid characterization of antibodies and has advanced their use for treating cancer and many other diseases.”

Giant soap bubbles

These methods have established Carol Robinson as a true pioneer in using mass spectrometry for analysing protein complexes. She has almost single-handedly founded a subfield of mass spectrometry proteomics, despite fierce criticism. This has required being fearless, innovative and creative. The latter became obvious when she decided to study some of the most challenging and important structures in biology – membrane proteins.

“Membrane proteins are incredibly hard to study, because one part of the protein exists inside an oily hydrophobic membrane, whereas the parts inside and outside of the cell are hydrophilic. We got the idea to coat them in detergent and then send them into the mass spectrometer in a giant soap bubble. And miraculously, this bubble shield really protects them, so they are released into the gas phase intact in a folded state.”

In a series of landmark studies, Carol Robinson unravelled the structure of the proteins synthesizing our cell’s energy currency, ATP, and how lipids play a key role in the structure and function of rotary ATPases, molecular motors involved in converting biological energy in our cells. Her more recent work on the role of lipids in membrane protein complexes has led her to study G protein–coupled receptors.

“These membrane proteins are targets for many drugs. We demonstrated that it was possible to maintain drug binding to G protein–coupled receptors and thereby identify natural ligands to these proteins. This enables new drugs to be identified that can bind to G protein–coupled receptors and thus target specific cellular processes.”

Freedom of movement

Carol Robinson’s group is now making exciting inroads into how fields related to mass spectrometry can dictate how drug discovery is performed. Once more, she has proved her critics wrong, who suggested that she had to use other techniques such as nuclear magnetic resonance or electron microscopy to study the structures of proteins.

“But if you think about a mass spectrometer, it is not in solution and it is not in a solid as a crystal would be, so it is not constrained. If you try to run through a swimming pool, it is really hard work. But if you want to express yourself, you want to be out in the air, so I think you could have your protein molecules in the gas phase rather than seeing it as a disadvantage.”

Her critics claimed that protein folding in a vacuum is madness, but Carol Robinson sees it as an advantage, because the proteins maximize their freedom of movement. They can express themselves, and something can be learned from that movement. However, as always, scientists need to accept that, even though they find their ideas exciting, others might not.

“Sadly, if you do something for the first time, a lot of people do not believe it. So there was criticism of the first experiments, because people said: ‘How can you measure folding in a mass spectrometer?’ I always wanted to be able to do something for human health, and everybody would say to me: ‘Well, that’s really never going to happen,’ but I think you just need to have the belief that it will.”

So most importantly – according to Carol Robinson – you need to believe in your own ideas and follow your passion.

“I have had a great career in science, but I like to think that anyone can do this. I want to dispel the myth that you have to be a genius. You need imagination and creativity. Drive and energy. These are the most important things.”

The 2019 Novozymes Prize was awarded at a ceremony on Friday, 15 March 2019 to Carol Robinson, Professor of Chemistry, University of Oxford.

Carol Robinson
Doctor Lee’s Professor of Chemistry
Carol Robinson holds the Chair of Doctor Lee’s Professor of Chemistry at the University of Oxford and is a Dame Commander of the Order of the British Empire. Carol is the first female Professor of Chemistry at the University of Oxford and was previously the first female Professor of Chemistry at the University of Cambridge. She is renowned for pioneering the use of mass spectrometry as an analytical tool and for her ground-breaking research into the 3D structure of proteins. Carol was a graduate student at Churchill College from 1980 -1982, completing her PhD in two years. Following an eight-year career break to begin raising her three children, she returned to research at Oxford, later becoming a titular professor in 1999. In 2001 she returned to Cambridge to continue her research into mass spectrometry and was elected a Professorial Fellow at Churchill College, a Fellow of the Royal Society in 2004, and a Royal Society Research Professor in 2006. In 2009 she was elected Doctor Lee’s Professor of Chemistry at the University of Oxford and in 2013 was awarded the title of Dame Commander of the Order of the British Empire.