Anal. Chem. 2008, 80, 7918–7920
IMS/MS: its time has come Rajendrani Mukhopadhyay The technique is expected to become more popular now that a commercial instrument is available. Folks cannot help but get a bit giddy with excitement when discussing ion mobility spectrometry coupled to MS (IMS/MS). “IMS/MS is finally here,” states Michael Bowers of the University of California Santa Barbara. “I think it’s going to be the dominant and new mass-spectrometer-oriented technology of the next 3 or 4 years.” IMS/MS has begun a new chapter. Since the 1960s, researchers have built IMS/MS instruments on their own, a practice that limited the field to specialists. Research with IMS/MS largely focused on atomic clusters and molecules like fullerenes in the first few decades of the technique’s use. In the 1990s, work by several groups demonstrated that the technique could be applied to larger and more complex biological molecules. But, despite its promise in biological investigations, IMS/MS remained in the hands of experts who had enough technical know-how to build their own instruments. “It is a tricky, complicated mass spectrometer to build,” says David Clemmer of Indiana University. All that changed in 2006 when Waters Corp. introduced a commercial, stand-alone instrument called the Synapt High Definition MS (HDMS) system that brought the method to the doorsteps of nonspecialists. The increased visibility of IMS/MS in a commercial format has produced a surge in applications, mostly in biological endeavors: proteomics, lipidomics, wholeprotein analysis, small-molecule investigations, and tissue imaging. “I’ve been just stunned,” says Clemmer of the breadth of applications that IMS/MS has found. It’s even become useful for polymer characterizations. Alan Millar of Waters explains that biopharmaceutical companies have begun to use the technique to analyze polyethylene glycol (PEG), a polymer that gets attached to biotherapeutics to increase the stability of the active compounds in vivo. “But the actual characterization of that PEG can be extremely challenging,” explains Millar, because PEG can be polymerized to varying lengths and branches. Biopharmaceutical researchers “thought IMS/MS could offer them a way to get a more detailed and rapid characterization of PEG. That has certainly proven to be the case,” he says. 7918
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This article will discuss the Synapt instrument as well as Thermo Fisher Scientific’s field asymmetric IMS (FAIMS) interface, which can be coupled to a variety of mass spectrometers. Table 1 lists vendors who make customized IMS/MS instruments. This article is not intended to be an exhaustive analysis of all IMS/ MS instruments; interested readers should contact the vendors for more information about their products and services. WHAT IMS/MS HAS TO OFFER IMS instruments separate ions on the basis of size, mass, and charge and are heavily used by security and military forces for the detection of explosives, narcotics, and chemical warfare agents (Anal. Chem. 2003, 75, 435 A-439 A). In IMS/MS, ions are separated on the size-to-charge ratio in the IMS component and the mass-to-charge ratio in the MS component. “By coupling them, you get a two-dimensional separation based on size and mass,” explains Herbert Hill at Washington State University. “You’re able to separate isomers or conformers of different compounds.” The ability to isolate conformers and isomers by IMS/MS affects a number of biological research areas, which the vendors are 10.1021/ac8018608 2008 American Chemical Society Published on Web 10/31/2008
Table 1. Companies that provide customized IMS/MS instruments.a Company
Excellims Corp. 978-264-1980 www.excellims.com Contact vendor for quote
Ionwerks 713-522-9880 www.ionwerks.com 750,000
Tofwerk AG +41-33-511-1151 www.tofwerk.com 350,000
Typical cost of service (U.S.D.) Type of mass spectrometer Standard quadrupole; other configurations Orthogonal TOF Orthogonal TOF; high-resolution or coupled to ion mobility available by agreement compact versions available component Type of ion mobility Standard high-resolution or chiral Standard (periodic focusing at Standard, high-resolution, and spectrometer 2-10 Torr He) atmospheric-pressure available Additional information Standard ESI and ambient-pressure, Instrument is specially designed Can couple to LC; ion sources can be high-resolution ion mobility spectrometer are for MALDI and MALDI/ion electrospray or corona discharge; interfaced to quadrupole mass spectrometer mobility imaging ion mobility spectrometers are via linear interface; optional orthogonal resistive glass tubes from Photonis with ion extraction interface available for resolution of 100-120; the instrument superior ion transportation efficiency; has been designed in collaboration also offers a novel chiral ion mobility with Herbert Hill at Washington State spectrometer/quadrupole mass University spectrometer for rapid separation and identification of enantiomers a
Contact vendors for more details about instruments and services.
targeting. “Because of the growth in MS-based proteomics, I think commercial companies realized that they could sell IMS/MS as a variant of LC/MS,” says Perdita Barran at the University of Edinburgh (U.K.). For instance, often in proteomics, peptides of similar masses but different charges can wind up as a single peak in a mass spectrum. But with IMS/MS, peptides of similar masses can be separated from a complex mixture on the basis of their charge state. The analysis of protein assemblies by IMS/MS has proved to be fruitful. Brandon Ruotolo, a research associate in Carol Robinson’s laboratory at the University of Cambridge (U.K.), explains that researchers have attempted to tease out information about protein assemblies by MS/MS. “The basic idea was you would take a protein assembly composed of many different protein constituents. You would put the assembly into the gas phase, and you would try to break it apart inside the instrument,” he says. “The idea was you were going to try and figure out which protein was connected to what based on the pattern of dissociation products you saw in the instrument.” Unfortunately, it was very difficult to predict the size and shape of proteins on the basis of a mass spectrum. But with IMS/MS, the size, shape, and mass information are available. Now “if we put the contact maps together with the sizes of each of the [protein] piecessand we use the pieces of information to constrain modelingswe can build 3D models, rather than 2D contact maps of protein complexes,” says Ruotolo. Researchers can also analyze the different conformations and dynamics of intact proteins with IMS/MS. Unlike the static and stiff images seen in textbooks, proteins are active, flexible entities. Researchers are interested in learning how these shifts in conformation come about. But MS only gives mass information, losing all the information on shape. IMS/MS provides a handle on how stretched or collapsed the proteins of interest are, a parameter known as the orientationally averaged collision cross section. And the separation of ions by shape and size before massbased separation comes with a practical advantage. Hill says, “A reason why the commercial instrument [by Waters] is successful is that it separates out the random noise into the mobility space, so it actually decreases the noise around the [m/z] peak. It can increase the sensitivity as well.”
HOW IMS/MS WORKS Almost any type of mass spectrometer can be interfaced with an ion mobility spectrometer, but popular choices tend to be TOF, quadrupole, and ion trap mass spectrometers. The Synapt HDMS system has a TOF mass spectrometer. The advantage of coupling a TOF mass spectrometer to an ion mobility spectrometer is speed. “On a regular IMS system, you scan at 50 Hz. You get 50 spectra/s,” explains Hill. “In a TOF mass spectrometer, you can get 50 kHz. You’re getting 1000 spectra per IMS spectrum, so it’s fast enough to take a full mass spectrum of the IMS spectrum.” Traditional IMS. It’s important to appreciate that different types of IMS exist. Drift tube IMS is the traditional form that has ions moving through a homogeneous, continuous electric field in a drift tube in the presence of neutral gas molecules. The time it takes the ions to travel down the drift tube is directly proportional to how big they are in space. Bowers uses skiing to explain the concept: “If you were skiing with your arms wideopen and standing straight up, you present a bigger cross section to the air molecules that are going to slow you down, rather than when you’re skiing in a tucked position. You’re still the same you, but yet you go slower when you present a bigger cross section.” The fundamentals are so well-known that from the time it takes an ion to traverse the drift tube, investigators can fairly accurately calculate the orientationally averaged collision cross section of the ion down to a few angstroms. Traveling-wave (T-wave) IMS. The second type of IMS is based on “traveling waves” and is the version used in the Waters instrument. Here, a series of low-voltage waves push the ions down the drift tube after they emerge from an electrospray ion source. The ions are propelled through a gas-filled ion guide. As the ions are pushed by the low-voltage waves, they are separated by sizessmall ions collide less frequently with gas molecules and make it first to the mass spectrometer, and large ions collide more and get delayed in their journey. A second T-wave device, operating at lower pressure, pumps all the ions into the TOF mass spectrometer so sample loss at the interface of the parts is reduced. So far, though, researchers can’t calculate the shape of an ion on the basis of the time it takes for the ion to travel through the drift tube. An algebraic expression to relate T-wave drift time to an ion’s collision cross section is still being developed. Experts Analytical Chemistry, Vol. 80, No. 21, November 1, 2008
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say this is one of the current challenges in working with the Synapt HDMS instrument. And because researchers can’t plug travel time in a drift tube into an equation to get an ion’s size and shape, the instrument has to be calibrated against a traditional IMS instrument. “In order to get cross-sectional information, we take standards that have been measured on research-grade drift tube instruments, and we measure those same standards in terms of their drift time on the Synapt,” explains Ruotolo. “We assign them a cross section based on these drift tube measurements. We can then use that information to calibrate the time axis to a cross-section axis.” Ruotolo says he and his colleagues calibrate the instrument every time they use it because ion mobility in traditional form can be affected by parameters such as fluctuations in temperature and small variations in pressure inside the drift tube. “These are things important to measure to as many significant figures as you possibly can in order to get the most accurate measurements,” he says. FAIMS. Although other types of IMS exist, T-wave IMS and traditional IMS in combination with MS are discussed most frequently in the literature. FAIMS works differently from traditional and T-wave IMS because it involves an asymmetric electric field. FAIMS has been commercially available since 2001 when it was marketed by a company called Ionalytics. In 2005, Thermo Fisher Scientific acquired the company and began to sell the FAIMS component under its own name. The FAIMS component can interface ion trap, quadrupole, or Orbitrap mass spectrometers. The electric field oscillates inside a FAIMS device so that ions of a certain kind can travel down the path while others hit the sides and don’t reach the mass spectrometer. “A traditional ion mobility instrument works on separating molecules on the basis of some physical property such as shape or size. It separates them linearly in time,” explains Julie Horner of Thermo Fisher Scientific. “FAIMS is a little twist on that, in that the separation is based on the difference in ion mobility when a low field is applied as opposed to a high field. It is ion mobility, but it’s not separating them linearly in time. FAIMS acts as an ion mobility filter instead of an ion mobility analyzer.” In other words, a FAIMS device separates ions on the basis of how much their mobility changes as a function of field strengthsnot size. The user can adjust the rate at which ions come out of the ion mobility separator or even “park” the device on a specific part of the separation. This feature of FAIMS reduces the need for fast mass spectrometers (like a TOF) that can keep up with the millisecond separations achieved routinely by drift tube IMS systems. FAIMS currently is geared toward certain specific uses. “FAIMS is really best suited for removing chemical interferences, doing impurity analysis, and so on. It rejects a large excess of something undesired in favor of transmitting something you do want to see,” says Horner. Its filtering capability helps researchers in proteomics and pharmaceutical analysis to clear out chemical noise in samples. A significant challenge in FAIMS is that nobody has yet figured out how to tie the data derived by the technique to a structurally descriptive parameter, such as size. T-wave and drift tube IMS are nice that way, says Ruotolo, because users obtain a physical parameter that describes the structure of the ion. “In fact, no one 7920
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that I’ve talked to can universally describe the FAIMS separation as tied to one physical parameter of an ion, like size,” he says. “People have tried to use ideas like ‘flexibility’ or ‘reaction chemistry with the background gas’ as a single descriptor for all ions, but neither holds up under scrutiny. The search is still on!” But as the instrument gains more traction in the market, an understanding of how it works and of its potential applications will develop further. “As [the technique] gets more present in people’s minds, they are looking at it as a separation, almost like gas-phase chromatography,” says Horner. MORE EXCITING THINGS TO COME? Experts are convinced that the use of IMS/MS will grow into more areas. “Once the proteomics community is more aware of the advantages [of IMS/MS], it will especially help with label-free proteomics,” says Barran. “The more exciting thing to come will be to look at protein dynamicssto look at changes in conformation as a function of time and as ligands are added.” And she goes on to list small-molecule analysis, metabolomics, and personalized medicine as other potential applications for the method. Experts say imaging of tissues by IMS/MS is sure to catch on. “It looks at the spatial localization of compounds in tissue. The results are very visual and indicate in a slice of liver or brain tissue where a compound is,” says Millar. “The ion mobility [component] gives you another separation dimension and makes those pictures far more defined.” Bowers suggests another path into which the technique can branch. “I think it’s going to head into spectroscopy because you’re going to be able to shape-select conformers and then do spectroscopy on a conformer-selected species. You will really be able to pin down a lot of the detailed interactions in biomolecules that you can’t do any other way.” But, despite the heady optimism, experts acknowledge that some challenges remain. Calibration of the T-wave ion mobility spectrometer is one. Resolution is another. The Synapt HDMS may be a high-sensitivity instrument, but it suffers from low resolution. “By the very nature of the traveling-wave device that’s been developed for this instrument, it’s never going to be a highresolution instrument,” says Bowers. But he notes that new developments may lead to an instrument with both high sensitivity and resolution. Managing the data is a concern. Because IMS/MS provides information on shape, size, and mass dimensions, “all your data scale up in terms of the size of the data sets. If you really want to use the data in its fullest glory, all of the computing that goes along with analyzing the data needs to scale up,” says Clemmer. “We are really at the limit of what can be done with reasonable computers at the bench at this moment. Computing becomes a major obstacle.” But none of these things seem to be a deterrent. Experts say the commercial instruments have just broken ground on IMS/ MS, and more sophisticated instruments, along with new methodologies and applications, are sure to follow in the next few years. “I think we won’t recognize IMS/MS 5-6 years from now,” says Bowers. Rajendrani Mukhopadhyay is a senior associate editor of Analytical Chemistry.
AC8018608
