The first prototype ion mobility mass spectrometry (IM-MS) instruments were developed over 60 years ago and the first commercial instruments became available nearly 20 years ago.1 While this technique has not gained the same level of acceptance in separation science as many other techniques with similar timelines, this sentiment is shifting. IM-MS is being explored as a tool across a variety of fields, including environmental analysis, drug development, omics studies, and forensic science. This technology continues to advance rapidly, increasing interest as it opens up possibilities for a wide range of important applications that aren’t well served by other separations technologies.
Recent advances in IM-MS
The field of IM-MS has seen substantial growth over the past 5–10 years, owing to the introduction of new technologies and the explosion of applications for which IM-MS has demonstrated significant performance gains. Integrating ion mobility with mass spectrometry has traditionally been seen as a novelty technique that could solve some niche problems, but actually hindered many of the standard analytical workflows where mass spectrometry shines. However, this is rapidly changing as researchers demonstrate that the latest generation devices have overcome many of the limitations of early IM-MS systems. This has resulted in a revelation that ion mobility can increase the sensitivity, speed, and accuracy of mass spectrometry measurements. This is perhaps most visible in the field of proteomics, where Thermo Fisher Scientific’s field asymmetric ion mobility spectrometry (FAIMS) and Bruker’s trapped ion mobility spectrometry (TIMS) technologies are employed to deliver analyses where researchers can detect 100 times more proteins per hour or use 100 times less sample material than possible a decade ago on systems without ion mobility.2
It is now widely recognized that the most capable mass spectrometry systems rely on some form of ion mobility to achieve ultimate performance. In particular, structures for lossless ion manipulation (SLIM) is a new ion mobility technology that demonstrates the highest resolution, full range analysis of any ion mobility approach, including FAIMS and TIMS. Launched commercially in 2021, SLIM can deliver an ion mobility peak capacity 5–10 times greater than other IM approaches, exceeding 200. This is equivalent to a 30-minute liquid chromatography (LC) separation but occurs 1,000 times faster. Most importantly, the resolution and speed performance does not come at the expense of sensitivity, since SLIM provides near-lossless ion transmission and high-duty cycle operation. This type of fast, tunable separation prior to mass spectrometric detection is more analogous to a chromatographic technique than it is to existing ion mobility approaches and offers opportunities to leverage ion mobility in new and exciting ways.
Exploring the concepts of SLIM technology and HRIM
SLIM is a new methodology for controlling ion processing within analytical systems. It is a flexible design architecture that uses printed circuit board technology to create ion optical devices that can trap and transmit ions in an intermediate pressure range of approximately 1–4 torr. More specifically, this is accomplished by printing electrode patterns onto two mirror image circuit boards and then arranging these boards with an approximately 3 mm gap between them. Ions can then be passed between the boards, and voltages applied to the electrodes allow researchers to trap, separate, accumulate, redirect, filter, and manipulate ions to create sophisticated ion processing operations. In the context of ion mobility mass spectrometry, SLIM is an incredibly powerful framework for designing next-generation ion mobility devices that are more cost-effective, robust, and high-performance than alternative options. Since its invention by researchers at Pacific Northwest National Laboratory (PNNL) in 2014, SLIM has been integrated with more types of mass spectrometers than any other IM technology, including multiple models of triple quadrupole, Orbitrap, time of flight, and quadrupole time of flight systems. It has also been used to generate the highest resolution ion mobility measurements ever performed thanks to its ability to transmit ions multiple passes through serpentine paths on the circuit boards without ion loss.
SLIM achieves ion mobility separation using a technique known as traveling wave ion mobility spectrometry, where a phase-shifted waveform is applied to a repeating sequence of electrodes to create waves of electric field that propel ions through the device. Residual gas molecules between the boards create a drag force that opposes the motion of the ions, with larger ions experiencing a stronger resistance force. This means that ions of varying sizes or mobilities will travel at different speeds relative to the traveling wave electric field and take different amounts of time to pass through the separation region of the SLIM device. A typical SLIM separation is performed over a 13 m distance, and its resolving power—defined as the measured size of the ion in square Angstroms divided by the width of the measured peak—is usually above 250. It is this resolving power metric that defines which ion mobility techniques are able to provide high-resolution ion mobility (HRIM) measurements. HRIM is generally defined as greater than a resolving power of 100, and only a few techniques currently achieve this level of specificity. These include TIMS, cyclic TWIMS, and SLIM, though drift tube ion mobility spectrometry and FAIMS systems have also demonstrated HRIM performance with certain limitations. However, SLIM is the only ion mobility technology able to provide full spectrum HRIM-MS performance without stitching of composite data scans.
HRIM is a separations technique that allows us to directly probe the chemical structure of ions in a manner highly complementary to the typical measurement of atomic mass performed by a mass spectrometer. With HRIM, we can generate a parallel measurement of the size, shape, and charge of a molecule that, when paired with its measured mass-to-charge ratio (and potentially also a retention time index), creates a highly specific profile of parameters that can be used for identification. The most obvious use case for HRIM is for isomers or isobars that are not adequately resolved in the chromatographic or mass spectrometric dimension. HRIM can often provide an orthogonal approach to resolve interferences and enable more accurate identification and quantification of analytes. HRIM can also allow for the reduction or elimination of chromatographic separations, increase the speed of fragmentation analysis, enhance detection sensitivity, and provide myriad other benefits. Given the multifaceted ways ion mobility can enhance mass spectrometry measurements, the thoughtful application of IM-MS to existing and emerging workflows represents an exciting puzzle that is sure to continue enticing scientists to explore.
Existing applications of HRIM and SLIM
One industry that is perennially at the forefront of technology adoption and innovation is biopharma. We have seen significant interest in the capabilities of HRIM in the characterization and development of drug products and the discovery of druggable targets. Protein and peptide therapeutics are particularly challenging to characterize, and traditional analytical approaches that work for small molecule drugs are often unsuitable for these applications. In many cases, finding a means to quickly and accurately identify the drug product is of utmost importance, as that assay can be deployed to address needs throughout the development pipeline, including questions about its efficacy, metabolism, structure, formulation stability, and mechanism of action.
Cyclic peptides offer a particularly interesting application case since they represent a capabilities gap for most existing LC-MS approaches. Peptide therapeutics can be designed to target certain biological pathways but are often subject to hydrolytic or enzymatic metabolism. To extend the lifetime of the drug in the body to ensure it reaches its biological target before degrading, these peptides are often cyclized to minimize steric access to sensitive regions of the molecule. Ensuring proper sequence and structure of the engineered product requires a methodology to differentiate between forms that result from backbone cleavages at different locations along the ring. By chemical definition, each of these metabolite forms are isomeric and it is not possible to generate non-chimeric fragmentation spectra by mass isolation alone. With HRIM, it is often possible to resolve these isomeric forms in the ion mobility dimension and selectively fragment them, delivering clear evidence of the identity.
Another area where HRIM is blossoming is in the analysis of per- and polyfluoroalkyl substances (PFAS). This class of molecules has been a go-to for use in a range of industrial and consumer products, including examples such as nonstick cookware, dental floss, and flame retardants. The fact that these substances are often referred to as “forever chemicals” highlights their stable nature and persistence in the environment. The recent revelation that these compounds may have substantial environmental and biological impact has necessitated the development of tools to detect and monitor them. HRIM is especially suited to the analysis of these molecules, which feature highly branched structures and isomeric complexity. The multifaceted applications of SLIM to the analysis of PFAS include rapid screening, forensic fingerprint analyses, non-targeted analysis, toxicological assessments in blood, and even bioaccumulation studies. Without the specificity to characterize the various forms, efforts to characterize the effects they may be having on biological processes will be severely obscured and hindered. Additionally, there is a need to sensitively and accurately screen for and monitor these species in various environmental matrices of varying complexities, and ensuring accurate annotation and quantitation is nearly impossible without the use of HRIM. This analytical challenge is common to the producers of these products, the agencies looking to regulate them, and the researchers who want to better understand their behavior and impact.
Potential future applications for HRIM and SLIM
Researchers are just scratching the surface of what can be done with SLIM, so there are an incredible number of potential applications to pursue. A few that are perhaps most exciting are multi-omics analysis and LC-free workflows. Starting with multi-omics, where there is a desire to integrate the detection of multiple chemical classes into a single workflow as a fast, chemical agnostic separation technique, HRIM has a tremendous technical advantage. Current multi-omic approaches typically comprise multiple analytical workflows that are highly reliant on chromatography, which must be tuned to a particular chemistry. Multiple data files for each sample generated from these various analytical configurations and run conditions are then leveraged to create a composite picture of each sample. But wouldn’t it be better to capture peptide, protein, DNA, RNA, glycomic, lipidomic, and metabolomic data in a single run? It has long been understood that the mass spectrometer is indifferent to the type of ion it is analyzing, but the number of possible analytes present in complex samples necessitates a separation prior to MS analysis to remove detection ambiguity (and increase sensitivity). HRIM is tailor-made to address this challenge by providing full spectral analysis, transmitting and separating all ions for subsequent MS detection. There are, of course, still challenges in generating ions of all these types from a common sample, but the added class-agnostic specificity afforded by HRIM is sure to advance the boundary toward the ultimate goal of single-shot multi-omic characterization.
The other area where HRIM is destined to have a significant impact is for workflows that are not amenable to the time, complexity, or robustness issues associated with chromatographic separations. There have been numerous attempts to accelerate and simplify MS workflows by removing the LC. A short list of recent attempts includes the Sciex Echo acoustic ejection, Agilent RapidFire, Advion Open Port, Bruker/IonSense DART, and MassTech AP-MALDI sample introduction systems. These systems have been met with various levels of success depending on the complexity of the injected sample. All of these devices operate in a manner suitable for HRIM-MS analysis, and the significant boost in overall peak capacity from the HRIM separation is sure to reduce chimeric spectra and potentially unlock a new paradigm in complex sample analysis without the need for chromatography. Such a development would enable a 10–100 times increase in throughput for these types of samples overnight. Coupled with the growth of big data processing solutions and the need for population-scale statistical analyses, this could be a real game changer.
Key challenges in expanding the use of SLIM and HRIM
There are two main challenges that must continually be overcome to achieve broad adoption of SLIM and HRIM. The first is the learning curve that all researchers must traverse to understand how any new technique can be employed to benefit their work. Every day, I have conversations with people who rely on LC, MS, NMR, immunoassay, or some other technique to support their analytical needs, trying to understand their application and how we might translate the demonstrated capabilities of SLIM and HRIM to address their pain points. There is always a barrier to letting go of how we have done things in the past to embrace new solutions. In many cases, HRIM offers an entirely different way of approaching a problem, incorporating new measurement principles, different data types, and novel parameters to optimize. Relying less on LC, for example, when those skills and methods have taken decades to develop can be difficult. There is a burden of learning new material and becoming proficient in translating its application to the particular analytical problem at hand before the benefit can be demonstrated and quantified. Only certain motivated individuals are willing to put in the required effort.
The other challenge area to be addressed is the process of converting the raw data into answers. While not unique to SLIM and HRIM, it is an ever-present struggle that we are generating data that reveals details about a sample that other techniques leave unseen. This means that we have new peaks in the data that aren’t reported in the literature and for which there is often no purified standard to confirm identity. To build up this knowledge base and enable confident identifications, there is a reliance on expertise from the researcher to interpret and translate the meaning from the raw data. At MOBILion, we have the task of not only helping to develop the tools to make the measurement but also the software to assist researchers in understanding the data. This entails making software intuitive and easy to use as well as capturing the knowledge from cutting-edge research to help researchers become successful. While it is sometimes a daunting task, it is certainly a worthwhile challenge to try to revolutionize separation science with high-resolution ion mobility.
Daniel DeBord, PhD, is the Chief Technology Officer for MOBILion Systems, Inc. He leverages his 15 years of experience developing novel analytical instrumentation to address challenges across a range of application spaces. For the past 10 years, DeBord’s work has focused on developing ion mobility technologies, including trapped ion mobility spectrometry (TIMS) and structures for lossless ion manipulation (SLIM), and exploring how these new techniques can be coupled to mass spectrometry instruments to access higher performance in fields such as proteomics and biopharma characterization.
[1] May and McLean, Anal. Chem., 87, 3, 1422–1436, 2014. https://doi.org/10.1021/ac504720m
[2] Peters-Clarke T, Coon J, Riley N. Instrumentation at the leading edge of proteomics. ChemRxiv. 2023; doi:10.26434/chemrxiv-2023-8l72m
This article is featured in our June 2024 publication, Innovation and Sustainability in Modern Analysis. Find out about the latest innovations and sustainable advances in mass spectrometry, chromatography, and related techniques.
