A new CCS calibration methodology and how it benefits the TWIMS community

We spoke with Brandon Ruotolo, Professor of Chemistry at the University of Michigan, and Keith Richardson, Principal Research Scientist at Waters, about their collaboration on a newly developed calibration approach and what it means for the future of travelling wave ion mobility spectrometry (TWIMS).

Read on to hear the process of their development method and how it will impact IMS research.

The development path of a new CCS calibration method

Collision cross section (CCS) values are an extremely useful experimental parameter, providing structural information and assisting in the identification of compounds in TWIMS experiments. However, the intricate motion of particles in TWIMS experiments means that ion mobility transit times must be calibrated to enable accurate CCS measurements. Traditional calibration techniques for accurately determining CCS values in TWIMS experiments function well for singly-charged species, and for multiply-charged species under more restricted instrument conditions. Also, for large multiply-charged species it has historically been necessary to select appropriate calibrants for different analytes. An improved CCS calibration method was sought that could simplify the process and expand the reach without sacrificing accuracy or precision.

How did Waters and the University of Michigan begin collaborating on TWIMS calibration research?

Brandon: My laboratory, and specifically, Sugyan M. Dixit, a graduate student in my laboratory, was interested in driving forward TWIMS theory. We approached Keith with questions about a trapping phenomenon in TWIMS ion guides and from there, discussed a new calibration approach that sparked his interest. It was clear from the initial conversation that collaboratively, we could develop an innovative set of equations and a theoretical description of the velocity of ions in TWIMS ion guides into a form that could be used as a practical calibration structure.

With that aim, we collected an enormous 20,000 calibrated data sets for the project!  The Herculean undertaking ranged from simulations to ion transport equations that were remarkable in terms of their creativity and insight. It was through the efforts of folks like Sugyan and David Langridge of Waters research that it was put into practice to enable data generation. Altogether, it’s been a real joy to work with Keith and the rest of the Waters team on this project.

Keith: I would absolutely echo that, Brandon. Our team had some ideas about calibration; we knew that there were important physical effects that could be incorporated into calibration, but it was also clear that it would be very challenging for one group to acquire the volume of data needed, especially as there were many different conditions to test to validate the approach. Instead, we approached it together as a group, and it worked nicely. With Brandon’s extensive knowledge of TWIMS and structural biology, we were able to ensure that the approach we developed would be relevant to the community, and that the methodology would be rigorous and robust enough for widespread use.

Could the improved CCS calibration approach affect IMS research?                                       

Brandon:

I would say our new calibration approach takes all of the rules around traditional CCS calibration and renders them essentially obsolete.

With this new approach, you can set your instruments to run under any optimized operating conditions and expect high-quality CCS data. Additionally, you can use a simple calibrant mixture, comprised of just two chemical species, to calibrate a wide range of ions, independent of charge states, cross section or chemical class. In principle, you can even house that calibration mixture in a reference sprayer for convenience and rapid calibration. The software package that we’ve developed to deploy this also really supports ease of use for the community. Altogether, I think the main effects will be a dramatic increase in the throughput and precision of TWIMS CCS, which could lead to breakthroughs in medicine, food science, environmental sciences, biophysics, and other fields.

Keith: Building on that, from our point of view measurement of CCS values and m/z values should simply be intrinsic capabilities of TWIMS instruments; the user shouldn’t have to think much about how to make a calibration or how to tailor the calibration to a given experiment. CCS calibration should be as straightforward and routine as mass calibration.

Ensuring that scientists can use this calibration approach across all our IMS enabled instrumentation was also a goal, and one which we successfully achieved. By allowing methods developed on one Waters TWIMS device to be transferred easily to another, users can be confident that they will acquire similar quality results from all of them.

How has the calibration method impacted your own research?

Brandon: The biggest impact for my own research group has been the precision with which we can assign CCS values to proteins and protein complexes. One area my group is interested in is assaying the structures of proteins and complexes that are involved in a wide range of human diseases. In many cases, the structural changes that are important in the etiology of those diseases are small. The greater the precision you can assign to a CCS value, the greater the power that value has to define the structure of that biomolecule.

Where we’ve seen this benefit manifested most strongly in my laboratory is in an alternative form of IMS analysis, which we call collision induced unfolding (CIU). This experiment involves taking a protein in the gas phase, heating it, and unfolding it during the measurement. When a protein unfolds in the gas phase, it tends to generate a wide range of intermediate structures that all have their own CCS. We treat these data as fingerprints to work out the details of a structure. So, if you can define each of these transitions with greater precision in cross sectional space, then that leads to greater and more differentiating fingerprints for important structural conformations in the context of our disease-based analysis. I think this can be applied to any laboratory interested in using these types of measurements for researching structural biology or biophysics.

Moving forward, how do you see CCS calibration techniques evolving?

Brandon: In the future, I think the basic ideas we put forward in this new calibration approach will begin to be more completely evaluated on next-generation TWIMS devices like the SELECT SERIES Cyclic IMS system. We’ll begin to see higher resolution, more precise CCS values generated by these devices that go well beyond the precision, accuracy, and resolution of the calibrants that we use. I believe that transformative technologies like the Cyclic IMS will cause us to reevaluate the calibrants that we use for measurements, because they were in many cases generated on technology that’s now two or three generations out of date.

I think developing very accurate primary standards for CCS calibration is going to be a community-wide effort, and one that’s definitely worth doing. We are just beginning to realize the power of CCS for applications and in metabolomics and proteomics. Omics applications rely upon our ability to get very precise CCS values, and I think our collaboration is definitely part of that journey.

Keith: As Brandon says, even on the current or previous generation of instrumentation, we can see that the precision of the instrument is greater than the accuracy of the calibrants we have access to. So, having access to more accurately measured reference standards in future will be a benefit not just to us, but also to the wider ion mobility community. We may also see the calibration process become ever more automated and routine in future, perhaps with instruments that effectively set up and qualify themselves. I think that would make it more accessible for a wider range of users.

Along with the previously described CCSonDemand, the innovative CCS calibration software has been approached through collaboration as part of a developing Micro-Apps strategy in Waters Strategic Engineering and is aimed at delivering innovative software more quickly with enhanced focus on customer needs.

Would you like to learn more about the TWIMS CCS calibration approach? Click here for more information, or here to try out the software.