CD-MS: Closer Than You Think

When did CD-MS first enter your radar?
 

I had been admiring Martin Jarrold’s work on CD-MS for years. However, it seemed out of reach for us to do technically, and our applications were not quite ready for it. On the application side, we first got interested in CD-MS through our work on native MS of nanodiscs. We had been working on carefully crafting mixed lipid nanodiscs that were still resolvable by native MS. When we saw that it was possible to do CD-MS on our existing instrument, we had to give it a try. Our first application was with nanodiscs made from natural lipids that could not be resolved by conventional native MS. It was exciting to see the mass distribution of the heterogeneous nanodiscs and watch this distribution shift as we added membrane-binding peptides.

Being able to do CD-MS on the instrument we already had in the lab was incredible. We did not have the expertise or resources to build a custom instrument, but it was relatively easy to get started by simply retuning the instrument.

What are the most promising applications of CD-MS today?
 

The most well-developed application is obviously in AAV capsids, where a lot of work has been done already. However, I think there are a lot of promising applications in biopharma with challenging new products, such as polymer-protein conjugates. I also think there are more fundamental applications to large complexes extracted from living systems with minimal sample preparation.

Although CD-MS has extended mass spectrometry into the mega- (and giga-) Dalton range, there’s talk of pushing beyond this into even larger particles. But for the things we are interested in, I do not see the need to go further than we can now.

What are the current limitations of CD-MS?
 

I think there are a lot of opportunities for better sample preparation and introduction. We are especially excited about coupling separations with CD-MS to help resolve highly heterogeneous samples. The challenge with separations is that CD-MS can be too slow to get enough scans in the available chromatographic elution time. My lab has been working to develop multiplexed injections to resolve this duty cycle mismatch. By stacking together multiple injections in a Hadamard sequence, we can preserve retention time information while dramatically increasing the number of ions detected. I think we will see more at the boundary of separations and CD-MS as people work to speed up CD-MS data collection to match chromatographic time scales and people work to interface innovative separation strategies with CD-MS detection.

What does the commercial landscape for CD-MS look like?
 

The DMT mode on the Q-Exactive UHMR from Thermo provides a commercial CD-MS option that is available now. I know commercial instruments from other vendors are also coming down the road. It is an exciting time to watch the technology develop.

I think the main barriers remain the need for expensive instrumentation, careful sample preparation, and experts to run the system and analyze the data. As instrumentation becomes more widely available, sample preparation methods improve, and more people are trained in the technique, it will continue to spread more widely. We have been working on the data analysis side to develop open-source software for CD-MS analysis, and I hope that has helped lower the barrier to entry.

What is your overall assessment of the current state of CD-MS?
 

Having done analytical ultracentrifugation (AUC), I think CD-MS is much easier. It is faster and requires a lot less sample. On the other hand, mass photometry is even simpler and easier than CD-MS, and I think it is better for simple applications. Where CD-MS will continue to expand is in more challenging applications where the unique capabilities of CD-MS can stand out. Of all these techniques, CD-MS provides the best potential resolution and unique abilities to interface with separations and multistage measurements like MS/MS and ion mobility. I think we will see more and more applications that combine CD-MS with other analytical methods.