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PNNL Team Boosts Performance of SLIM Ion Mobility System


NEW YORK – Recent advances by researchers at Pacific Northwest National Laboratory could significantly boost the power of their structures for lossless ion manipulations (SLIM) ion mobility separation system.

Detailed in a pair of papers (here and here) published in Analytical Chemistry, the advances include an approach that allows for more efficient utilization of analyte ions, improving mass spec sensitivity as well as a multi-level SLIM device that allows for extremely long ion mobility paths and very high resolution separations.

Ion mobility uses differences in size, shape, and charge to separate ions in the gas phase. When coupled to mass spectrometry it gives an additional level of separation beyond that provided by liquid chromatography, allowing researchers to achieve better coverage of the molecules in their samples or distinguish between highly similar analytes like isomers that are difficult for conventional workflows to deal with.

All the major life science mass spec vendors have incorporated versions of the technology into their instruments in recent years. Agilent Technologies has partnered with MobiLion Systems to integrate the PNNL SLIM technology with its mass spectrometers. Chadds Ford, Pennsylvania-based MobiLion has the exclusive license to the technology from PNNL.

The PNNL device uses SLIM technology to extend ion mobility path lengths beyond that allowed by conventional IMS systems, potentially enabling much more extensive separations. 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 without increasing the footprint of the device.

The separating ability of an IMS system increases with the square root of the path length, meaning that that the longer an IMS path, the greater the device's resolving power. One way the PNNL researchers have sought to further expand the SLIM system's resolving power is by routing ions through the path multiple times, like laps around a track.

This comes with the problem, though, that ions with higher mobility overtake and lap ions with lower mobility during their multiple cycles through the path, which could create overlapping peaks that could complicate analysis and counter the device's separating power.

To get around that problem while still keeping the device instrument relatively compact, the PNNL team has developed a system using stacked sets of SLIM paths with ions moving up to the next level after they finished the route on the lower level.

In the Analytical Chemistry paper, the PNNL team presented a four-level SLIM device that provides a total path length of 43.2 meters. They provided a proof of principle using the system to resolve a complex mixture of 42 heavy labeled phosphopeptides and found that while many of these phosphopeptides remained unresolved following a pass through a single-level SLIM system, the four-level system provided much higher resolution, managing even to separate out phosphopeptide isomers that were identical except that they had phosphates modifying different serine residues.

Richard Smith, director of proteomics research at PNNL and one of the leaders of the IMS development effort, said the group is now planning to build a 10-level SLIM system.

He noted that as the system adds more and more levels, the heat produced by the system begins to present challenges as high temperatures can affect the behavior of the molecules in the system and impact the reproducibility of the measurements.

This issue could be addressed by changing the materials used to build the SLIM devices, which currently use "conventional printed circuit board technology," Smith said. "So there are practical issues, but the fundamentals of how to move ions between different levels, it works well and makes sense."

The group's other advance concerns how the device is able to accumulate ions prior to running them through the IMS system.

In electrospray ionization (ESI), the most common type of ionization used in proteomics research, ions are generated for analysis by applying high voltage to them as they come off the LC column. These ions must be collected and trapped in the IMS system prior to separation by the device, which Smith said has traditionally presented challenges due to the fact that the devices can only store a limited amount of ions.

"Typically this has been done in what we call an ion funnel trap, and [that trap] limits you to about a million charges," he said.

The SLIM system is able to devote a larger space to trapping and so is able to accumulate a much larger collection of ions for analysis. However, typically, the system has not been able to accumulate ions while running them, which means that while it is separating a packet of ions, the ions being continuously generated by the ESI source are not being collected by the IMS device and are therefore not analyzed, decreasing the sensitivity of the mass spec experiments. This is particularly an issue for SLIM as the long path lengths enabled by the technology take longer to run than in conventional IMS systems.

To get around this problem, the PNNL team devised a SLIM system that decouples the ion accumulation and separation regions, allowing it to simultaneously collect and separate ions.

"That means we can basically double the duty cycle and make use of all the ions," Smith said. "We increase the ion utilization efficiency from around 50 percent to close to 100 percent."

He noted that this ability to capture and measure all the ions produced was particularly important for the work on single cells or other small samples he and his colleagues were pursuing using SLIM coupled to single-cell proteomic technologies developed at PNNL.

"It's a real advantage because we can trap those ions and accumulate as many as we can create and then measure them all," he said.

The SLIM system also provides an advantage for single-cell proteomic workflows in that it provides researchers with additional information they can use to better identify peptides at the MS1 level, meaning they don't have to use two rounds of fragmentation, which provides more confident identifications but at the cost of sensitivity.

Mobilion CEO Melissa Sherman declined to say whether the company was incorporating either of the advances published by the PNNL into its systems but noted that the company has exclusive access to PNNL's SLIM IP portfolio and that it considered the "flexibility and tunability and customization" of the SLIM device "one of the significant benefits of the technology."