Expert Insight: Why is ion mobility mass spectrometry becoming the must have tool in life sciences research?

Watch this on-demand webinar to learn about ion mobility mass spectrometry and its applications in life sciences

05 Aug 2020

Prof. Konstantinos Thalassinos, University College London, and Prof. Ian Wilson, Imperial College, London

The coupling of ion mobility with mass spectrometry has developed into a powerful and widely used separation technique with applications in many research areas across the biological sciences. In this webinar, Prof. Ian Wilson, from Imperial College London, and Prof. Kostas Thalassinos, from University College London, highlight the benefits of IM-MS for two different application areas.

Probing the structure and dynamics of proteins by means of a cyclic ion mobility spectrometry traveling wave device. Prof. Thalassinos will demonstrate how traveling wave cyclic ion mobility enables the detailed study of proteins and protein complexes without significantly affecting their structure and how tandem ion mobility experiments allow the study of protein behavior not observable using other techniques.

Ion mobility MS enabled metabolic and lipid profiling. Prof. Wilson describes how the inclusion of ion mobility within rapid metabolic and lipid phenotyping workflows increases throughput and provides cleaner data to aid database searching, and how calculated and measured collision cross-section values can be compared to increase confidence in the identification of potential biomarkers.

Read on for highlights from the live Q&A session or register to watch the webinar at any time that suits you.

Q: You showed that to get comprehensive coverage, you need to use several different systems. Is there a specific order that you would try them in?

IW: You cannot try them all simultaneously unless you've got a lot of instruments. I would generally start with reverse phase chromatography in positive ion mode. If that showed some interesting things, I'd move straight onto negative ion and then move to HILIC, but it does depend on the context of what you're trying to do. So, if I wanted to get a full phenotype, that would be the process. If I already had a hypothesis that was interested in polar metabolites, then I'd go to HILIC first. So, it depends on what you're trying to do, but certainly, if you have no idea what you're trying to do, which is where I usually start from, I'd go from reverse phase.

Q: How important are software developments, and what feature developments do you think will be needed to make the most of cyclic data?

KT: The software is now becoming the bottleneck, not just for the cyclic that I just showed here, but the whole of the mass spec community and field because the instruments are getting faster and more sensitive. As it showed you with a cyclic, we are getting this extra layer of information. This is important and therefore we've started developing some software to tackle this. I think more so for tools to investigate the depth of this data, but also to try to apply new machine learning approaches that are becoming very popular now. I don't know whether we can apply deep learning to mine this data. I think it's certainly the case, but we need to look into this and I think the other thing in the native mass spec and mobility field that we are now trying to address is also coming up with new file formats, open file formats, in order to disseminate the data, which will further allow computational people to go in and look into this. The community has come together and we are working on that.

Q: What are the biggest advantages of microbial?

IW: The biggest advantage of going to microbial for me is that providing you've set the instrument up properly, the separations are as good as you get from using 2.1-millimeter columns, but you're able to use a lot less sample or the same amount of sample and get much more sensitivity. In my experience, I never have enough samples to do all things I want to do. That's a big advantage and there are other advantages as well. There's a significant reduction in mobile phase usage, and that's a good saving both in cash because good HPLC grade solvents are very expensive and if you generate a lot of waste solvent, you may have to pay for its disposal. So, using a microbial column is both good for you in cash terms and for the environment. 

Q: Are CCS values reproducible across multiple sites?

IW: In the paper that was highlighted a little earlier in the "Journal of Chromatography," a "Comparison of collision cross-section values obtained in traveling wave ion mobility spectrometry," we did look at the same set of standards across three different sites in two continents, and they were very close. Of course, you have to ensure that everything's been set up properly and everybody's using the same conditions. I think they probably are reproducible, and even if they're not as reproducible as one would like this would be getting better all the time. The advantages in this field are very rapid indeed. So, I think they are a good guideline and with the calculation, the ability to calculate CCS values, I think that will be another real advance for us as well.

Q: You showed great applications of cyclic full probing protein conformation. What other application molecules do you think will benefit from the cyclic and the slice CA methodology?

KT: The cyclic instrument is very versatile. The ability to do all these different kinds of experiments and manipulate your ions opens the possibility for analyzing a wide variety of molecules. So, I think it will have a good impact in every type of molecule. For our purposes, I think we see, we started doing some experiments in looking at glycans, both into how the glycans affect the protein structure, but also into actually looking at the glycans themselves. They’re quite complicated, the connectivity, and you’ve got different isomers. I think these kinds of experiments are going to have a big impact here.

Q: Is it necessary to check the metabolites in both positive and negative ion modes?

IW: In one of my earlier slides, I started to use the Donald Rumsfeld phrase, I ‘misunderestimated’ the time it takes to analyze a sample, because although I was talking about using reverse phase chromatography and HILIC and it could take up to 30 minutes to analyze those samples, in fact, it takes a bit longer than that because you do have to use both positive and negative, I think, if you want to be certain you're seeing everything and that probably includes HILIC as well. How I'd qualify that is if your test and control samples are identical in positive ion reverse phase, I wouldn't expect them to be distinguished by negative ion or HILIC either. Now, that's an opinion rather than a fact. So, if you have the time and the instrumentation, I would look in both positive and negative, and in HILIC and in reverse phase. If you have a limited amount of instrumentation, I would start on positive ion reverse phase and see what I get and, if I get nothing, I probably wouldn't go forward.

Q: Since some diseases are associated with the normal conformation of their native protein structure, is it possible with this advanced technique to at least state the structure of that altered or normal protein in the diseased states?

KT: We're interested in looking at these proteins that are involved in misfolding in aggregation. It depends on what we talk about with structure. Ion mobility is a very low rate technique so we're not going to get for that domestic structure. If we combine it though with molecular dynamics calculations, we might start assigning some of these intermediates that we are seeing and that's something that has been done in the field and we now want to do for these increased features that we see from this, like CM methodology. So, I think that's going to be important future work. The key benefit I see here is also seeing how the different conformers change upon conditions. For example, if we find a small link and we stabilize some of the more compact conformers or do we promote more extended conformers to appear? So, I think that's where the ion mobility is very good. At the moment, we are looking at these differences and how it affects the individual conformers.

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