Capturing proteins by mass spectrometry as they go about their business in complexes may provide vital clues about how they work.
Capturing proteins by mass spectrometry as they go about their business in complexes may provide vital clues about how they work, Carol Robinson tells Cath O’Driscoll
Mass spectrometry has come a long way since Carol Robinson started work at Pfizer in the 1970s. But then so has Robinson herself. Now professor of mass spectrometry at Cambridge University, Robinson left school at 16 to work as a technician at Pfizer. It was while there that she was first introduced to the technique: ’Mass spectrometry was really taking off in organic chemistry. I was involved at a very early time and it was very exciting’, she recalls.
Recognising her potential, one of her supervisors at Pfizer advised her to ’get some more qualifications’ and, with the company’s sponsorship, Robinson qualified as a Graduate of the Royal Society of Chemistry (GRSC) by doing day release at Medway and Maidstone College of Technology. Inspired by this experience, she went on to do a PhD with Dudley Williams at Cambridge, using mass spectrometry to study peptides and other relatively small molecules, followed by a year’s postdoctoral work at Bristol.
After that, however, Robinson had other plans. It was to be eight years - and three children - before she went back to the laboratory. The opportunity to return came after a visit to the local library with the children; while there Robinson spotted an ad for a mass spectrometrist required at the University of Oxford. It was a fairly junior position, which was ’tough to go back to’, but what really surprised Robinson was just how much mass spectrometry had changed while she’d been away: ’During my career break that’s when electrospray MS took off. So I missed the early times and when I came back I was amazed at what could be done’.
Electrospray ionisation (ESI) was originally developed as a method for looking at polymers, producing multiply charged ions that brought the molecules down to a mass-to-charge (m/z) range where they could be measured by conventional mass spectrometry. By the early 1990s, when Robinson began work at Oxford, spectrometrists had started using the technique to study individual proteins. Researchers at Yale University in Connecticut had reported the first intact protein spectra in 1989.
Surprisingly, Robinson had few problems getting to grips with the technique: ’It was actually easier and more robust than the sort of MS that I had been used to’. She began using ESI-MS to study protein folding - specifically how secondary structure forms in the hen lysozyme protein. The idea was to incorporate deuterium into the protein’s secondary structure and monitor the change in mass as a function of folding time. ’I remember the initial excitement caused by the observation of three distinct peaks [corresponding to the unfolded, partly folded and fully folded protein] in those spectra’, Robinson recalls.
Even in those early days, however, Robinson always had a bigger goal in sight: protein complexes. Large protein complexes are at the heart of all major biochemical pathways and are the key to understanding how proteins carry out their multiplicity of functions. Robinson started looking at small protein-ligand complexes held together by non-covalent bonds. The fact that most of these complexes - involving various cofactors and molecular chaperones - typically dissociate in the mass spectrometer meant that she and her coworkers were able to study complexes otherwise inaccessible by MS techniques.
But this ability came at a price: while the group was able to study individual components, there was no way of telling how many of each of these components was present, in other words the stoichiometry of the original complex was unknown.
Then in 1996 Robinson and her coworkers hit upon a surprising observation. Looking at the mass spectrum of a protein-coenzyme A (CoA) complex, some of the peaks clearly corresponded to the mass of the whole complex. This was not the first protein complex known to remain intact under MS conditions, but the fact that the group had come upon this observation almost by accident prompted them to study the structure of the complex in more detail.
Its ’survival’, the team realised, was due to the fact that it is held together by ionic interactions which are strengthened in MS conditions, unlike hydrophobic interactions which are weakened. Armed with this information they quickly went on to probe the interactions in many more protein-ligand complexes.
Using the standard quadrupole spectrometers available at that time brought serious limitations, however. Restricted to studying compounds with a m/z ratio of no more than 4000, researchers could apply the technique to complexes involving only a few proteins. The vast majority of useful and important complexes include many more.
The solution that spectroscopists eventually hit upon was to couple ESI-MS with time-of-flight analysers, which should in theory allow access to even the largest macromolecular complexes. In 1999 Robinson applied this technique to obtain a mass spectrum of the intact ribosome - with a molecular weight of ca 2.5MDa this is still one of the largest particles for which mass spectra have so far been recorded. Ribosomes are the protein synthesis factories of living cells. The E. coli ribosome analysed by Robinson contains over 50 different proteins and three large RNA molecules.
To carry out this analysis, Robinson and the team collaborated with equipment supplier Micromass to develop a powerful new mass spectrometer capable of operating at m/z ratios well above 100,000. (This type of spectrometer generally has a m/z limit of 16,000.) The trick, Robinson says, ’was to reduce the radiofrequency of the quadrupole mass filter and introduce collisional cooling into the system’ - preventing the compounds from dissociating during transfer from the solution to the gas phase. Robinson is confident that the group will be able to extend the technique to even higher masses; one of her current collaborations with researchers at the University of California at San Fransisco involves the 10MDa protein complex of a virus.
But the technique does eventually start to show limitations: ’We can go higher and higher in mass, but what is limiting is the homogeneity of the complex. More and more small molecules bind to the larger ones, which means that we can’t really resolve anything. You end up with a broad m/z envelope rather than being able to measure the species... You’re losing the point when you start to get really high masses’.
The reason that MS is so important for looking at complexes is stoichiometry, Robinson says: ’you can tell exactly the mass and hence the composition, and that gives you the number of each type of protein component in a complex’. In principle, X-ray crystallography could tell you the same information. But obtaining the necessary crystals to carry out the analysis is often tricky. ’Some of these complexes are dynamic and heterogeneous so they don’t crystallise readily’, Robinson elaborates.
MS also provides complementary information to X-ray; it allows researchers to see regions of proteins otherwise too dynamic and heterogeneous for other techniques to access. ’We can’t actually match the amazing detail that X-ray crystallography has, but there are regions, for example in ribosomes, that are not observed in the X-ray analysis but which we can study using MS.’
One other previously elusive region is the so-called stalk structure of ribosomes. Projecting from the main ’core’ of the ribosome complex, the stalk is very dynamic in solution. Pinning down the structure by MS, however, Robinson and coworkers are able to observe a pentameric complex that changes, for example, in the presence of elongation factors involved in protein synthesis.
How exactly these elongation factors interact with ribosomes is a matter of some controversy among scientists working in this area. Robinson’s latest data may shed light on the issue: ’We have seen some peaks that we think might be the nascent peptide chain within the ribosome - that’s quite exciting if it’s right. It would give us an opportunity to look at protein synthesis in action’.
Not all of the complexes that Robinson is interested in are quite so huge. Transthyretin is a 56kDa tetrameric protein complex associated with the disease transthyretin amyloidosis. One of the causes of this disease is deposits of amyloid fibrils similar to those seen in the more common Alzheimer’s disease. These form when the normally soluble transthyretin protein comes out of solution and deposits the insoluble fibrils in organs such as the heart, liver or kidneys.
If they could stop these fibrils from forming, Robinson and coworkers hope to be able to find a way of inhibiting the disease. In collaboration with Jeff Kelly at the Scripps Research Institute in California, the group has recently identified several small molecules that bind to and stabilise the transthyretin complex, thereby preventing it from coming out of solution.
Taking a lead from nature, many of these small molecules prepared by Kelly and his coworkers are analogues of transthyretin’s natural binding partner, the hormone thyroxine. Two small molecule ligands bind to each transthyretin complex and in competitive binding assays with thyroxine several molecules compared favourably. ’It was a blind trial so we didn’t know what to expect. We ranked the ligands according to their efficacy in stabilising the tetramer’, Robinson says.
An equally important factor in deciding which of these small molecules may be useful is ’to know that the complex maintains its ability for vitamin A transport in the presence of the synthetic ligand’, she adds. This ability to ferry vitamin A about the body is a normal function of transthyretin, and also involves binding to a second protein, retinol binding protein. Several promising ligands that bind to transthyretin, without affecting its ability to bind to retinol binding protein, are now in further tests.
An even more important target for new drug development is Alzheimer’s disease. An estimated 12m people currently suffer from the disease worldwide and thousands more are predicted to succumb as the world’s population steadily ages over the coming years. Would the small molecule inhibitors Robinson and coworkers have identified for transthyretin have any effect on the fibril formation in the brain seen in Alzheimer’s disease? Based on the outcome of early studies, it seems not, Robinson concludes: ’Ideally we were hoping that we would find similarities with transthyretin amyloidosis. Most of the ligands we have are specific for transthyretin, but the same processes that we have used to study ligand binding to transthyretin would also be appropriate for Alzheimer’s disease’.
As the human genome continues to yield up its secrets, even more protein targets associated with disease start to emerge. For proteomic researchers seeking to identify each of these individual molecules, MS is one of the workhorse tools of choice. More useful yet, Robinson believes, will be the power of MS techniques for characterising protein complexes. ’When we got our first high mass complexes we were very excited because you started to think about how far it could take you. We are now combining it with proteomic strategies to look at ex-vivo complexes which are unknown’.
They are not the only protein hunters to go after complexes. Two recent papers in the journal Nature reported the use of MS to ’go fishing’ for large multi-subunit protein complexes. ’One of the drawbacks was that they couldn’t tell how many of each individual protein was in these complexes. What they did was fished for a complex and identified the different types of protein present by MS, but to know how many you’d need to keep the complex together and weigh the whole thing’, Robinson says. That’s where Robinson is hoping that her approach will have the edge. Since moving to Cambridge last year she and the group’s collaborators have been establishing a suitable target on which to try out these ideas. With such a vast number of complexes to choose from, small wonder that this is where Robinson has her sights set on the future.
Source: Chemistry in Britain
Carol Robinson recently won the RSC’s award for mass spectrometry for 2001, sponsored by Thermo Finnigan.
As its name implies, electrospray is the process of generating a fine mist of sample droplets by applying an electrical current or voltage across a sample. Since the 19th century, industry has exploited this process for applying paints and other coatings to metal surfaces.
Electrospray ionisation works in a similar way, by generating charged ions ready for analysis by mass spectrometry. In the most common set up, a liquid sample is pumped into a sharply pointed hollow metal tube, such as a syringe needle (see Fig). A high voltage power supply connected to the tube outlet charges the liquid at the needle tip, from which a jet of similarly charged liquid droplets is expelled. The liquid droplets in the jet repel each other as they travel away from the tip, breaking up to form a plume or mist of fine droplets that evaporate to leave behind the highly charged analyte molecules for analysis.
The attraction of electrospray for large biological molecules such as proteins and DNA lies in the fact that it produces multiply charged ions with little or no fragmentation. Electrospray ionisation is a soft ionisation method that does not require heat or a high vacuum, instead using pressure to deliver the appropriate flow of liquid through the needle. Unlike more conventional ionisation methods, which result in a wide variety of molecular fragments and singly charged ions, the molecule being ionised remains relatively intact during the process.