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New PNNL Ion Mobility System Could Replace LC in Some Proteomics Workflows


NEW YORK (GenomeWeb) – Researchers from the Pacific Northwest National Laboratory (PNNL) have developed an ion mobility separations (IMS) device that could allow some mass spec-based proteomics experiments to eliminate upfront liquid chromatography separations.

Described in a study published last month in Analytical Chemistry, the device uses structures for lossless ion manipulations (SLIM) technology to extend ion mobility path lengths, allowing for much more extensive separations than conventional IMS systems, said Richard Smith, director of proteomics research at PNNL and senior author on the paper.

He added that he expected that within the next year his lab would be able to use IMS systems similar to that presented in the study to replace conventional LC upfront of various mass spec workflows.

He also said that a company had been formed to commercialize the technology, though he noted that this process was still in the very early stages, and he declined to say who was leading this commercial effort.

Ion mobility uses differences in size, shape, and charge to separate ions in the gas phase. Mass spec vendors including Waters, Agilent, Sciex, and Bruker offer instruments incorporating IMS devices, which, in proteomics, researchers typically use to provide an additional layer of separation after conventional LC.

Adoption of IMS has grown in recent years, but, as the Analytical Chemistry authors note, these methods typically come with some drawbacks. One major challenge has been the ion losses associated with various IMS techniques, which lead to reduced sensitivity.

The SLIM technology used in the PNNL device allows researchers to manipulate ions without suffering losses. These SLIM systems use arrays of printed electrodes to confine ions within the ion mobility field. They also make it possible to route ions around turns without losses, meaning that an IMS drift path can be designed to run along a serpentine path, greatly increasing the length of the IMS path (and, therefore, the potential resolution of the separation) without increasing the footprint of the device.

For instance, Smith said, the IMS system described in the Analytical Chemistry paper is roughly 13.5 meters in length but fits in a space of around 1.5 square feet. He added that he and his colleagues have recently developed what they are calling an "ion elevator," which allows them to move ions between stacked sets of serpentine IMS paths.

"We've demonstrated this already over a couple of different levels, and without any ion loss," he said. "So, we can stack a large number of these — as many as we want, really — to build a much longer path length device, and still keep a compact design."

He said that this sort of stacked design could enable IMS devices around half a kilometer in length.

The device used in the Analytical Chemistry paper also contained an ion switch that enabled the researchers at the end of the IMS run to either direct the ions into the mass spectrometer or back through the IMS system for additional separation. In the study they demonstrated they were able to send ions through the system for as many as 81 passes (1.1 km in total length) without seeing a decrease in signal intensity.

One potential issue with this approach, however, is peak lapping, as ions with higher mobility overtake and lap ions with lower mobility during their multiple cycles through the path. This, the authors noted, could create overlapping peaks that could complicate analysis and counter the device's separating power.

Smith said that he and his colleagues were working on techniques for compressing peaks that will mitigate this issue. He noted, as well, that the sort of stacked device using the "ion elevator" approach would also get around this limitation.

The PNNL researchers used the device in the study to analyze two human milk oligosaccharide isomers, lacto-N-302 hexaose (LNH) and lacto-N-neohexaose (LNnH), which other IMS systems typically struggle to separate. At one pass through the instrument, the two species were resolved, and at nine passes the researchers were able to detect a new feature of LNnH that had gone undetected in previous IMS experiments.

The power of PNNL's SLIM SUPER (structures for lossless ion manipulations serpentine ultralong path with extended routing) system means the technology could allow researchers in proteomics and other fields to eliminate the LC separations typically done upfront of mass spec analysis, Smith said.

Were this to come to pass in proteomics, it would be a major advance for the field, as LC contributes significantly to the complexity and relatively low throughput of both research and clinical mass spec workflows.

Among the many factors limiting clinical adoption of mass spec for targeted protein quantitation is the time required for LC prior to mass spec analysis. To get good enough sample separation to enable quantification of the low abundance proteins often targeted by such assays, gradients of 30 minutes or more are commonly needed, which limits the number of samples that can be run by an instrument in a given day.

Clinicians and researchers have and are exploring a variety of ways to address this issue, including use of antibody enrichment of targeted peptides to reduce the amount of separation needed and use of mass spec methods like MALDI that don't require upfront LC. The SLIM SUPER system could represent another way to speed up such clinical assays, Smith said.

It could also up the throughput of the shotgun-style assays more commonly used in the research setting, he said. For instance, researchers often achieve deep proteome coverage through fractionation in which they divide a sample into dozens or more fractions, each of which they run separately. This means analysis of a given sample could require 50 separate LC runs lasting an hour to several hours each.

"So, in practice, we may take several days of instrument time to get deep coverage on a sample," Smith said. Replacing LC with IMS would significantly cut down on this time.

"For a lot of applications, [IMS] will be enough," he said "We get very high separation power. Right now we're very close to the peak capacity or separation power that you typically get with liquid chromatography. And, of course, it's a lot faster. The separations take a few seconds as opposed to maybe an hour with liquid chromatography."

PNNL has developed IMS technology used by Agilent in some of its mass spec instruments, and Smith said that he has discussed the SLIM SUPER technology with several mas spec vendors. But, he said, the new company that has formed to commercialize the technology has no relationship to existing vendors. He added that neither he nor PNNL are involved beyond licensing the technology.