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As FT/MS Matures, PNNL Instrument Rises to the Challenge of Throughput

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When it comes to Fourier transform mass spectrometry, the seemingly neverending debate over the merits of various mass spectrometers for proteomics applications has in the past boiled down to a familiar argument: While FT/MS is clearly the leader in terms of sensitivity, resolution, and mass accuracy for identifying proteins from complex samples, all that comes at a cost, in terms of price, complexity of analysis, and throughput.

But that argument may not hold up much longer. Thermo Finnigan, Micromass, and Bruker Daltonics are all taking steps towards bringing FT/MS into the mainstream, and recent advances at the Pacific Northwest National Lab promise to finally transform FT/MS into a high-throughput technique. Late this summer, Dick Smith and his team at PNNL, together with Bruker, assembled a prototype Fourier transform ion cyclotron resonance mass spectrometry (FTICR) system they say rivals more conventional techniques in sample throughput, ease-of-use, and robustness.

“The basic challenge and the reputation this kind of technology has is that it’s not high-thr throughput and that it's difficult to operate,” Smith said. “Our intention here was to demonstrate that it can be a robust technology, operate routinely, and produce data in a high-throughput fashion.”

Smith, whose lab also holds a home-built FTICR mass spectrometer operating at a magnetic field strength of 11.5 Tesla, set out earlier this year to transform a commercially available Bruker Fourier transform mass spectrometer operating at 9.4 Tesla into a souped-up FTICR instrument complete with automated operation and a front-end peptide separation system using liquid chromatography.

To do this, Smith’s team ripped out half of the hardware that accompanied the commercially available Bruker FT/MS system, replacing it with design elements developed at PNNL, including a high-pressure capillary liquid chromatography system and hardware for automating the interface between the LC and the mass spectrometer.

“The way we’re approaching this is to look at complete proteomes,” Smith said. “At the current state-of-the-art we’re doing a proteome characterization in about three hours, and we have the capability with this instrumentation to make about seven of these measurements per day.”

There are caveats, however. Although it is possible to analyze a proteome within this time frame, it’s likely to take considerable time and effort to set up the parameters for running an experiment at such high-throughput, said Don Hunt, a protein mass spectrometrist at the University of Virginia and consultant for MDS Proteomics and Thermo Finnigan. Referring to Smith’s recent study of the Deinococcus radiodurans proteome, published last month in PNAS, Hunt said that it appeared Smith’s team devoted considerable effort towards verifying their peptide mass assignments.

“It was an impressive accomplishment, and if he had to run that measurement over again it would probably only take him an hour to go through the whole thing,” Hunt said. “On the other hand, if he’s looking at different proteomes every three hours, that I think is based on considerable [prior] work.”

And while no FT/MS instrument capable of high-throughput analysis is currently available commercially, Hunt said his customized FTICR spectrometer, built around a 7 Tesla magnet, can analyze about 100,000 peptides in less than an hour.

But Hunt praised Smith and Bruker for apparently addressing the limitations inherent to FT/MS that restrict the number of ions that can simultaneously inhabit the mass analyzer. Because an FTICR mass spectrometer calculates an ion’s mass based on its frequency inside the analyzer, too many ions in the analyzer can cause space charge effects that distort individual frequencies. “We all know how to solve that problem, it’s basically by controlling the number of ions that go into your analyzer,” Hunt added. “If Dick has solved that in a convenient way, and he can do this reproducibly on a fast time scale, I would say that’s a major accomplishment.”

Hunt also pointed to Smith’s advances in developing techniques for accurate mass measurement of peptides, which allow the identification of individual peptides without having to resort to MS/MS analysis to determine a peptide’s amino acid sequence. Hunt’s group currently does not perform this type of analysis, and he said the accurate mass measurement technique represents “a real breakthrough” because it can also speed up database searches.

Which Vendor Will Be First?

Meanwhile, mass spec vendors are off to the races. Thermo Finnigan announced at Pittcon last March that it is developing a hybrid ion trap Fourier transform mass spectrometer that it hopes to unveil at next year’s Pittcon meeting, and Bruker hopes to begin selling its instrument based on the prototype in Smith’s lab in the first quarter of next year. Micromass also announced last month that it had joined forces with FT/MS mass spectrometer manufacturer IonSpec to build a multiple quadrupole FT/MS instrument that it plans to launch in Q1 of 2003.

While vendors are reticent concerning specifics about their new mass spectrometers, it is clear there will be differences in the magnetic field strengths associated with the various FT/MS instruments – as well as in their price. According to John Wronka, Bruker’s vice president and general manager for life science systems, Bruker’s instrument will be available at field strengths of both 9.4 Tesla and 12 Tesla, at a cost ranging from about $1.2 million to $1.5 million.

Hunt said the field strength associated with Thermo Finnigan’s instrument will “not be that high,” but the price will fluctuate around $650,000, according to Nelson Cook, Thermo Finnigan’s director of marketing.

And the response from the market towards the next generation FT/MS instruments? Wronka said Bruker has already taken orders.

– JSM

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