Bench philosophy: Ion Mobility Mass Spectrometry
by Steven Buckingham, Labtimes 03/2012
Figuring out interactions of proteins and protein complexes is a challenging task. Soft ionization mass spectrometry combined with ion mobility spectrometry may help to get the job done.
Electrospray ionization source.
Today’s need is to figure out not just how individual proteins work but how proteins interact with each other. After all, the big deal with proteins is that they have immensely complex shapes and the significance of this, in turn, opens up infinite possibilities for precise interactions with other proteins. Proteins are team players but understanding protein complexes is, well, complex. Part of the problem is that we are trying to understand massive (at least from a chemist’s view), dynamic structures and our traditional techniques just aren’t up to the job. In the meantime, the genomics data is ‘log-jamming’ up and the physiologists are impatiently tapping their feet...
But things are about to change. The log-jam is being freed up, if only a little, by the coming together of three important developments. Actually, the first two of these came together some while ago when mass spectrometry and ion mobility joined forces. But Ion Mobility Mass Spectrometry (IMMS) really took off, when in the 1990s B. Ganem and J.D. Henion discovered, that it is possible to preserve non-covalent interactions between proteins in the gas phase (Biol Mass Spectrom., 1994; 23(5):272-6).
The reason why structural biologists are so excited about IMMS is because it allows them to figure out not only what proteins are present in a protein complex but also which one binds with which, how strong their interactions are, what the overall shape of the protein complex is, and more. Perhaps even the atomic resolution model of the whole complex.
So how does it work? IMMS is, as its name suggests, Ion Mobility (IM) combined with Mass Spectrometry (MS). Forgotten what IM is? Let me remind you. The idea is that every ion or any small, charged particle, for that matter, travelling through a medium will tend to bump into things. Imagine, for example, a charged molecule travelling through a gas. You apply an electric field and that tends to push the ion through the gas. But occasionally, the molecule will hit an atom of the gas, slowing it down. It is a bit like trying to walk into a factory at the end of a shift.
Obviously, the more gas atoms, the slower the molecule will go and the more charged the molecule, the faster it will go. But of course there is also the effect of the molecule’s size – larger means more collisions, which means slower progress. So, if you measure the speed of the molecule as it works its way through the gas, you can figure out something about its charge and its size, although you won’t be able to separate the two. For a lot of applications this doesn’t matter – you may just want to spot molecules by their signature. When you last went through a major airport, there was probably an IM tube sniffing you for drugs or explosives, looking for the charge/size signature of known target molecules.
In distinction to IM, MS exploits the fact that molecules have a characteristic charge-to-mass ratio. So, put the molecules into a vacuum, charge them up and accelerate them with an electric field; the spectrum you get reflects the charge/mass ratio of the components. With IM you get the size/charge ratio, with MS you get the charge/mass ratio – put the two together and you can resolve size, shape, charge and mass.
Putting IM and MS together isn’t what is causing all the excitement. The third ingredient relates to the question of how to get all this to work with protein complexes. Okay, the technique works marvellously well for small molecules and has been doing so for some years. In fact, the first IMMS experiments date back to the middle of the 1980s. But before we start looking at protein complexes there is a major hurdle to overcome. That hurdle is about keeping the delicate structure intact enough for it to survive the ionization process and its flight through the IMMS tube. For the whole approach to work at all, you have to take your protein complex out of its nice, cosy, aqueous environment, place it into a gas (i.e. vaporise it) and, in the process, charge it up. Solve that problem and all the potentialities of IMMS open up.
It was the invention of nanoflow electrospray ionization (NEI) that solved the problem. How does NEI work? First, you introduce your protein-containing solution into a capillary. The solution works its way along the capillary and forms a tiny droplet at the tip. Then, charge up this droplet. Charge it up a lot. As the charge accumulates, the meniscus grows outward, forming what are known as “Taylor cones”. These cones release highly-charged droplets into the air, where their small diameter creates a large surface area to volume ratio. This means that the solvent evaporates rapidly, causing the droplets to shrink. But note what happens next: as they shrink, charge remains conserved, forcing like charges ever-closer to their neighbours. The coulombic forces of repulsion increase as the surface area decreases, resulting eventually in the droplet breaking apart. As the process continues, you are finally left with highly-charged protein complexes literally hanging in mid-air. The great point of all that is this: the process is actually very gentle from the molecule’s point of view and there are several reports that the non-covalent interactions, able to hold even quite large protein complexes together, survive it.
Being able to transform protein complexes into this state got some structural biologists into quite a state of excitement. In 2008, this excitement was fuelled by an impressive publication describing the structure of the eukaryotic translation factor, iEF3. There had been intense interest in this protein because of its central role in ribosome formation and protein transcription. But iEF3 is quite a monster of a protein, consisting of 13 subunits. Carol Robinson’s group (University of Oxford, UK) described, in a special feature of PNAS, how they were able to work out, which subunits bound to which and how strong these bonds are (Zhou et al., Proc Natl Acad Sci U S A., 2008, 105(47):18139-44.)
To do this, they exploited the way, in which NEI preserves interactions coupled with the tendency of proteins to dissociate predictably, depending on the ionic strength of the medium. Changing the ionic strength (or perhaps pH) of a medium causes protein complexes to dissociate in a predictable way, depending on the nature of the forces that hold them together. So, you can break the complex down reliably into smaller subcomplexes. The important point here is that NEI has been shown to preserve these subcomplexes. What does this tell us? First, recall that the passage of the complex through the gas (the “drift time”) depends on the size of the molecule. But the complexes are not spheres – so the drift time also depends on the orientation of the molecule. This means that by presenting different subcomplexes and different charges, coupled with some mathematical and computing wizardry, you can figure out the 2D, and hence the 3D, profiles of the complex. Clever!
Another way of analysing the structure of protein complexes is to let them break down in the IMMS process itself. If you pass the complex through an inert gas, the collisions the protein makes with the gas molecules causes the complex to break apart. By controlling the charge and the gas concentration, you can get conditions just right to be able to study this dissociation in isolation of any other factors, including any solvent. We are only just beginning to understand the process but it would appear that the collision causes the protein subunits to unfold momentarily and, hence, dissociate from the complex. Charge floods into the now-available interaction surface, causing even more structural changes. This opens up interesting avenues for understanding the process of protein folding.
But for the structural biologist wanting to understand the interactions between subunits, the real excitement over IMMS comes from combining it with other techniques, such as molecular dynamics simulations and other variants of molecular modelling. In essence, IMMS provides structural constraints for these models.
There is in one sense nothing new about this, of course. Molecular modelling has long been constrained by the coordinates produced by NMR and Xray-crystallography (XRC). IMMS just adds another gun to the battery of integrative techniques. But there are many things that IMMS can do that the others cannot. NMR and XRC require proteins to be purified in large amounts, and there are many cases where the proteins cannot be purified without completely altering them structurally. But if we are to take our understanding beyond single proteins and into the realm of quaternary structure, NMR and XRC simply do not offer enough constraints. You can’t just plug in your primary sequence for tens of proteins and run them through an energy-minimising routine with the proteins free to dock wherever they want – there are simply far too many free parameters. And you almost certainly won’t be able to crystallise the proteins and use NMR and XRC to obtain your constraints that way because there are few complexes that would survive the harsh treatment needed for crystallization.
IMMS fills this gap by providing constraints not only to the overall structure of the complex but also to its stoichiometry and even the forces binding it together. IMMS is far from being high throughput, so will not fill the genomic throughput bottleneck. But it has a classic hallmark of a technique, or family of techniques, that will fuel a rush of discoveries in the realm of protein-protein interactions – it is highly flexible and allows a battery of techniques to be pipelined together. No doubt researchers will become imaginative in the way they string together IMMS with other bits of the experimental toolkit. So, if protein interactions are your thing, I would put “IMMS” on your PubMed search keyword list, right now.
Last Changed: 10.11.2012