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Warwick Team's FT-MS Phasing Solution Boosts Instruments' Resolution, Sensitivity, Mass Accuracy

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University of Warwick researchers have developed a broadband phase correction method that can significantly improve the performance of Fourier transform ion cyclotron resonance mass spectrometers.

The method should be applicable to spectra from all varieties of FT-MS machines, including Thermo Fisher Scientific Orbitrap instruments, and offers two-fold increases in resolution and mass accuracy as well as a roughly 40 percent improvement in signal-to-noise ratio, Peter O'Connor, professor of analytical chemistry at University of Warwick and one of the leaders of the effort, told ProteoMonitor.

The bump in performance comes from producing the Fourier transform spectrum in the absorption mode instead of magnitude mode, which has conventionally been used in FT-MS. While researchers have long known that absorption mode offered improved resolution and mass accuracy, problems with phase correction have prevented researchers from using this mode.

The Warwick team managed to solve the phase equation allowing for broadband phase correction, which enabled them to use spectra in absorption mode.

In a study published in Analytical Chemistry, the researchers detailed their solution and used it for top-down analysis of the protein calmodulin, demonstrating a 1.54-fold increase in signal-to-noise ratio and a 2.4-fold increase in mass accuracy, compared to conventional magnitude mode.

"Ultimately we could see an extra 10 percent more peaks that we could pull up out of the baseline and resolved, where [in magnitude mode] we couldn't," O'Connor said. "So it gives us a little more depth to the information that we can pull out of the same spectrum."

The researchers did the work on a Bruker solarix 12T FT-ICR instrument, and several Bruker scientists were co-authors on the paper. However, the company's participation was largely limited to "logistical support, [such as] how you crack their files, how you calibrate their spectra, things like that," O'Connor said.

He and his colleagues are "trying to get [Bruker] interested" in applying the technique to the company's instruments, he said, adding that the company is "definitely interested, but getting them interested on my [shorter] timescale is a little bit different."

Bruker did not reply to a request for comment on the work.

The technique can be applied to essentially any FT-MS spectra, O'Connor said, noting that his team has "shown that we can do it with old spectra from old instruments that [other researchers] have recorded and sent in to us."

"So it's not something that is restricted to this one instrument or this one pulse sequence or something like that," he said.

In fact, O'Connor said, Thermo Fisher has incorporated a version of the technique – which it calls "enhanced Fourier transformation," or eFT – into its most recent Orbitrap release, the Orbitrap Elite. At the 2011 American Society for Mass Spectrometry annual conference, a team of Thermo Fisher researchers led by Alexander Makarov, the company's director of global research for life sciences mass spectrometry and principal inventor of the Orbitrap, presented a poster on the method.

Using absorption mode in the Orbitrap is more straightforward than in a typical FT-MS instrument due to the way the ions are injected into the analyzer, O'Connor noted.

"The phase function [in an Orbitrap] is basically a constant, or at worst case a fairly shallow linear function," he said. "Whereas in our case it's a quadratic function."

Alan Marshall, one of the inventors of FT-ICR and director of the National High Magnetic Field Laboratory ICR Program at Florida State University, has also published on applying absorption mode in FT-MS, including a paper two years ago in Analytical Chemistry.

However, O'Connor said, Marshall's group hadn't published the details of its solution to the phase function.

"I wanted to be able to do it [too]; I wanted the advantages," he said. "So we had to go off and find our own way to solve the phase function."

O'Connor said his group had no intention of patenting the finding, noting that "it would be hard to make any such patent stick since the methodology is pretty well known – it's just the tricks that we used to solve the phase function that were pretty novel."

Within proteomics, the method will likely prove most useful for top-down work, O'Connor said.

"Absorption mode is going to help you no matter what spectrum you apply it to because of the reduction in the baseline noise that you see," he said, "but it's going to help most in places where you have really complex spectra with overlapping peak distributions. So, top-down [proteomics] … really any system where you have a lot of peaks in the same system."

"For peptides in a bottom-up proteomics mode, it’s going to make very little difference in terms of resolution and accuracy because you've got peaks that are already widely spaced and easily resolved anyway," he added. "It's still going to help, but it's not going to help hugely like [in top-down]."

Northwestern University professor Neil Kelleher – a leading top-down proteomics researcher – told ProteoMonitor that absorption mode FT-MS could be thought of as not only offering a jump in resolution, but speed as well.

"It's not just two-fold resolution," he said. "In FT-MS you can trade off resolution for speed, so you can go twice as fast" by forgoing the increase in resolution. And, in the case of Kelleher's research, which aims to perform high-throughput intact protein analysis on an LC timescale, speed is key (PM 11/4/2011).

"Top-down [proteomics] is a great application for this [method]," he said.

The development provides a potential boost for FT-ICR machines as new mass spec instruments, primarily the Orbitrap Elite, make inroads into the top-down proteomics field. FT-ICR machines – a category traditionally dominated by Bruker – have typically been the favorites for intact protein work, but the Orbitrap Elite has attracted interest from top-down researchers since its introduction last June (PM 7/1/2011).

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