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Cal Tech Team Highlights Potential of Single-Protein NEMS-MS as Proteomics Research Tool

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Researchers at the California Institute of Technology have completed a proof-of-principle study using nanoelectromechanical systems-based mass spectrometry, or NEMS-MS, to measure single proteins in real time.

The work, detailed in a paper published this week in Nature Nanotechnology, represents the first use of NEMS-MS for real-time detection of individual proteins and offers a glimpse of the technology's potential as a future tool for proteomics research, Michael Roukes, a Cal Tech researcher and leader of the study, told ProteoMonitor.

NEMS-MS uses nanoscale resonators for analyte detection, tracking the changes in resonant frequency that occur when molecules adsorb to the resonator surface. Because each resonator fits only one protein at a time, the devices measure analytes at an individual level. And, as opposed to conventional mass spec, the resonators make their measurements based solely on mass, not mass-to-charge ratio, eliminating the need for ionization of target molecules.

According to Roukes, the technology is also potentially capable of making measurements over a significantly larger dynamic range than conventional mass spectrometry.

"We have a five-year plan to [build a device comprising] a million [resonators]," Roukes said. "And with a million [resonators] we project we'll be able to span in 20 minutes' time a concentration dynamic range of 1011. With that concentration dynamic range, we can actually measure the complete plasma proteome."

As Roukes noted, were NEMS-MS to fulfill this promise, it would be a potentially "disruptive technology" for proteomics research. Conventional mass spectrometry's limited dynamic range has been a crucial stumbling block for the field, hindering detection and quantification of the low-abundance proteins that many researchers think could prove most useful as biomarkers.

Despite the technology's potential, however, researchers must overcome significant challenges to make it a useful tool for proteomics research. Most crucial is improving mass resolution, which in the case of the Cal Tech device was in the tens-of-kilodaltons range.

At that level of resolution, the researchers were able to distinguish between different isoforms of human IgM antibodies, but it would not be sufficient for distinguishing between smaller molecules.

"That mass resolution holds even if we go up to multiple megadalton particles," Roukes said. "So our resolving power can be quite high for large proteins, molecules, or complexes." However, the resolution will need to be significantly improved for the device to prove useful for measuring smaller proteins or features like post-translational modifications.

Improving resolution is "an ongoing area of technological development," Scott Kelber, a graduate student in Roukes' lab and author on the paper, told ProteoMonitor. "Currently we can only measure large proteins, but we are gradually … improving our mass resolution and eventually we [will be able to] measure smaller proteins and peptides and post-translational modifications."

Recently, another group demonstrated detection with single-Dalton resolution on an NEMS resonator, Roukes said, noting that this suggests "that there's nothing standing in the way of us doing the engineering to get down to the single-Dalton [level]."

That demonstration, however, was done on an NEMS device built with carbon nanotubes, which, Roukes said, "are not amenable to the large-scale integration crucial to building" the highly multiplexed devices his group envisions.

Since the recently published work, Roukes and his colleagues have built devices combining up to 20 resonators and plan to up that to several hundred in the next few months, Kelber said.

The researchers are also working on new techniques for delivering target molecules to the resonators, Roukes said. In the recent study they used both conventional MALDI and electrospray ionization simply because these existing techniques were conveniently at hand. However, they would ideally use methods "specifically tailored to neutral-based mass spectrometry," he said, noting that such a technique could provide advantages particularly in the analysis of fragile molecules that can fragment during ionization.

Noting that Roukes' team was the first she knew of to demonstrate single-molecule detection using NEMS-MS, Anja Boisen, a professor in the Technical University of Denmark's Department of Micro and Nanotechnology, told ProteoMonitor that she saw the study as "a proof of concept, and I hope more demonstrations will come."

She suggested that development of an easy-to-use system for delivering molecules to the resonators, as well as a more straightforward read-out system, would likely be key to driving wide use of an NEMS-MS device.

"The whole system needs to be less complex in terms of how the sensor is read out and how the molecules are delivered to the sensor," she said. "I see this as one unique setup that will take a lot of time to duplicate in other labs. For such a technology to be picked up [more broadly], some effort needs to be put into simplifying the setup and probably making the user interface more 'friendly'."

Boisen added that it would likely be "some years before this could be achieved... many years, maybe."

According to Roukes, the engineering challenges involved in producing an instrument suitable for proteomics research – while formidable enough for a small academic group like his – are relatively simple "compared to building a modern computer microprocessor." He added that as the researchers scale up the complexity of their device, they plan to more significantly enlist the resources of their collaborators at the Micro and Nanotechnology Innovation Cluster, MINATEC, in Grenoble, France.

Their MINATEC colleagues – several of whom were co-authors on the Nature Nanotechnology paper – have access to "a billion-dollar, state-of-the-art microelectronics foundry" that will be key as they pursue more complex devices, Roukes said, noting that the researchers plan to do their future "proof-of-concept and technology development [work] for upscaling [the devices] in collaboration with our colleagues in Grenoble."

He and his colleagues have no current collaborations with industry regarding the device, but, he said, they have been awarded two patents covering the NEMS-MS technology specifically as well as "numerous complementary patents on the underlying technology, and more in the works."

Because the technique doesn't require ionization of target molecules, NEMS-MS devices can do without the ion optics technology used in conventional mass spectrometers for focusing and directing ion beams for detection, and this, Kelber said, could make them simpler, less-expensive instruments.

Roukes noted that in building the device the researchers had intentionally limited themselves to "the palette of materials from which semiconductor electronics chips are made" to ensure they can be easily mass produced.

"If we can build one, we can build a million," he said. "There are no barriers to massively parallel production of these devices."

Instrument cost, Roukes added, would ultimately depend on the level of demand, but, he said, "it's completely feasible that this is something in the thousands of dollars or tens of thousands of dollars price range."

At that price point, he suggested, NEMS-MS could prove attractive as a point-of-care technology – "a standard instrument in every doctor's office" – as well as a common benchtop instrument for proteomics research.

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