Recent advances in trapped and traveling-wave systems, along with the development of new platforms such as structures for lossless ion manipulation (SLIM), have propelled ion mobility into the high-performance era. And with the emergence of new approaches such as parallel accumulation mobility aligned fragmentation (PAMAF), ion fragmentation records are being broken, and exciting applications in single cell – and even single organelle – analysis are opening up.
Here, in the second installment of our series of articles on The Coming of Age of IMS (see Part One, featuring Erin Baker), we speak with Daniel DeBord, Chief Technical Officer at MOBILion Systems, and Nikolai Slavov, Professor of Bioengineering at Northeastern University, to find out what's happening at the bleeding edge of ion mobility innovation – and what it will take to make the technique a true analytical mainstay.
Could you give us a general introduction to the role of ion mobility in analytical science?
Since the 1980s, there’s been a growing interest in coupling ion mobility with other types of analyzers such as mass spectrometers. Operating on a size-to-charge, rather than mass-to-charge ratio, it addresses a key blind spot of mass spectrometry. This becomes useful when investigating analytes with similar mass-to-charge values or fragmentation pathways, yet differ in their atomic arrangement structurally.
The resolution of these isomeric and isobaric compounds represent the most obvious capability of ion mobility. However, its utility extends more generally to improve the sensitivity, speed, and specificity of complex sample analysis. By combining ion mobility with mass spectrometry, the overall peak capacity of analysis can be increased, meaning more components of a sample mixture can be separated from one another – and, in turn, improved confidence in compound identification and quantification.
In life science research, where biological samples represent some of the most complex mixtures in analytical science, coupling ion mobility with LC-MS/MS regularly enables the detection and quantitation of thousands of proteins, metabolites, and other biological compounds – despite the complex matrices in which they reside. Because the principles of ion mobility are fully defined by gas conditions and electrical potentials, these separations are incredibly reproducible, and provide a measurement characteristic for analytes known as collision cross section (CCS). These CCS values outline where researchers can expect to find a given analyte on the separation scale, similar to an accurate mass measurement in HRMS.
Have there been any key developments, technologies or events that have shaped the evolution of ion mobility in recent years?
While drift tube ion mobility represents the original approach to analysis, several alternative technologies have also emerged over the last two decades. Each of these techniques exploit the same fundamental ion-neutral collision methodology, but by varying how the electric fields and gas flows are arranged, they’ve managed to extend performance in resolution, sensitivity, and speed of analysis. These new technologies have primarily appeared as additional analysis stages within HRMS systems to enhance platform capabilities.
In particular, Bruker’s trapped ion mobility spectrometry (TIMS) products in 2017, and the high-resolution traveling wave ion mobility systems from Waters (2019) and MOBILion (2021), have significantly propelled the field forward. With these systems came substantial improvements in mobility resolution, creating a new class of instruments with resolving powers exceeding 200, compared with 20 to 50 in previous generations. This has enabled researchers to resolve ever more challenging compounds such as isomeric lipids differing only by double bond positions or orientations, and peptide conformations indicative of higher order structure.
Along with improvements in raw performance, the range of methods for coupling ion mobility separation with mass spectrometry has expanded rapidly in recent years. Bruker’s parallel accumulation–serial fragmentation (PASEF) method, for example, uses TIMS separation to focus precursor ions in time, enabling more efficient fragmentation and detection by a QTOF. This approach significantly boosts the speed and sensitivity of ion fragmentation analysis. Similarly, Waters’ SELECT SERIES Cyclic IMS employs a circular ion path with trapping and fragmentation regions before and after the mobility stage. This allows multi-step experiments that can isolate, selectively accumulate, fragment, and further separate analytes to explore chemical structures with exceptional specificity.
As previously mentioned, the high-resolution ion mobility tech being commercialized by my company (MOBILion Systems) is the most recent entrant into this space. Our technology, known as structures for lossless ion manipulation (SLIM), is based around the concept of designing ion mobility devices with long, serpentine paths using printed circuit board technology. This has enabled us to achieve the highest mobility resolving powers to date, exceeding 1,800, while also delivering mobility peak capacities that are 5 to 10 times higher than other techniques – and on par with typical high performance liquid chromatography approaches. These technologies are symbolic of the steady progress IM-MS has made, potentially facilitating new types of analysis.
What new capabilities are you and your team exploring with SLIM?
I’m part of a talented team of scientists and engineers at MOBILion Systems, working to maximize the robustness and capability of SLIM technology. Our overall goal is to explore SLIM’s ability to solve real world problems encountered by researchers. More recently, we’ve been focusing on how we can exploit the high peak capacity and fast separation capabilities of SLIM to enhance mass spec fragmentation analysis for non-targeted workflows like discovery proteomics. We’ve seen some really encouraging results on our prototype systems, increasing the number of proteins we can detect by 10 to 25 times when leveraging ion mobility. In testing, we’ve also found that we can boost the sensitivity of the Agilent QTOF system we’re integrated with to the extent that we can detect thousands of proteins from individual cells, enabling single cell proteomics measurements.
Are there any instruments, techniques or developments in current use that you’re particularly excited about?
The PASEF approach implemented on Bruker’s timsTOF systems has demonstrated the potential for ion mobility to increase the speed and sensitivity of MS/MS analysis. However, we’ve recently observed that by further increasing mobility resolution and peak capacity, we can reduce – or even bypass entirely – the need for quadrupole mass isolation as part of ion fragmentation analysis. We call this approach parallel accumulation mobility aligned fragmentation (PAMAF). Operating as a filter, transmitting certain species while discarding others, the quadrupole commonly used for precursor ion isolation can reduce sensitivity when many precursors still need to be isolated and fragmented. With PAMAF, up to 100% of the ions can be transmitted, fragmented, and detected with high-resolution ion mobility separation. This spreads the precursors out temporally so they can be analyzed independently, rather than selectively filtering for each in sequence.
In addition to boosting the sensitivity of fragment detection, this approach also increases the speed with which precursors can be isolated and fragmented. Using PAMAF, we’ve been able to generate ion fragmentation spectra at up to 1,300 Hz – which we think might be the record! With the potential to overcome the long-standing tradeoff between the number of precursors that can be isolated and the time spent on each, we believe this approach represents a breakthrough in how MS/MS measurements have been performed over the past 50 years. This may mean that we’ve finally found a technique for unbiased, nontargeted analysis that delivers the same sensitivity as targeted MS/MS approaches.
How is ion mobility currently being combined with other techniques, such as mass spectrometry or chromatography, and what are the benefits of these hybrid approaches?
Ion mobility is now routinely incorporated with liquid chromatography and high-resolution mass spectrometry analysis. This leads to a number of benefits; including increased overall peak capacity for complex samples, increased detection sensitivity, reduced chemical background noise, fewer chimeric fragmentation spectra, faster ion fragmentation analysis, higher sample throughput, and the ability to leverage CCS as an additional molecular descriptor. These advantages have cemented LC-IM-MS as the go-to analytical platform for characterizing complex mixtures and challenging analytes.
What are the key steps required to overcome current challenges and make ion mobility a standard tool in analytical labs?
In my opinion, the field faces three key challenges: developing better data-processing tools for ion mobility, addressing the cost and complexity of IM-MS instrumentation, and maximizing the usefulness of CCS measurements. Because ion mobility is often coupled with other analytical techniques like LC-MS, the data structures are inherently more complex. In many cases, legacy LC-MS software has been developed with a lack of awareness of ion mobility separation and limited functionality – if any at all. To harness the full power of multi-dimensional separations leveraging ion mobility, we must expand functionality and awareness within the tools we rely on to translate raw data into analytical insights across applications in areas such as omics.
To date, the latest generation of ion mobility technologies has been limited to high-end research instruments. However, there are opportunities to extend their use beyond high-resolution platforms to fit-for-purpose, nominal-mass instruments that could support more routine workflows. Such embodiments would enable broader access to ion mobility’s capabilities, enhancing chromatography-free separations with improved reproducibility and ease of use, for example.
I also believe the field is just scratching the surface in terms of the inherent value of CCS for chemical identification. By addressing software implementation gaps and broadening hardware integration, CCS has the potential to become as routine as intact mass measurement for identity confirmation.
The Shape of Things to Come
With Nikolai Slavov
Could you tell us about some of the key application areas where ion mobility is having the greatest impact?
“Ion mobility contributes to three major categories of applications, understood intuitively in the context above. First, ion mobility allows confident detection and quantification of isobaric analytes with similar chromatographic properties. Second, it substantially increases the efficiency of ion utilization and thus sensitivity via advanced operating modes like PASEF and PAMAF, as described above. These gains make even the most demanding applications, such as single cell, and even single organelle, analysis now feasible. Naturally, ion mobility-enabled instruments such as the timsUltra AIP are advancing the sensitivity limits of single-cell proteomic analysis. Within this context of sensitive analysis, ion mobility addresses another challenge: it removes singly charged contaminant ions, which can be abundant relative to peptide ions and would otherwise contaminate the mass spectra if co-fragmented. Finally, ion mobility supports increased specificity and depth of coverage of complex mixtures, such as mammalian proteomes. These benefits are particularly pronounced when using short separation times.
What’s your view on the long-term potential of ion mobility over the next 5 to 10 years?
“Modern science poses multiple demanding objectives for mass spectrometry. Namely, the comprehensive analysis of highly complex mixtures with an ever growing demand for higher throughput and sensitivity. While these objectives present major challenges for traditional approaches to deep proteome analysis, such as multidimensional liquid phase separations, ion mobility offers a promising solution. It provides the specificity and depth of multidimensional separations without the drawbacks of longer analysis times and higher sample requirements. Thus, in my view, ion mobility is ideally positioned to reconcile the demanding objectives of high-throughput, sensitivity and depth.
In future, I expect ion mobility to become an integral part of all high-performance LC-MS/MS systems used for comprehensive analysis. Technologies that improve the resolving power of separations, such as SLIM, and methods for efficient ion utilization, such as PAMAF and slice-PASEF, will propel IM-driven advances, providing continuous gains in sensitivity and throughput while increasing depth.”
Daniel DeBord is Chief Technology Officer of MOBILion Systems, Inc., USA
Nikolai Slavov is a Professor of Bioengineering and Founding Director of the Parallel Squared Technology Institute at Northeastern University, USA
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