This story originally ran on July 21.
Researchers at the ETH Zurich Institute of Molecular Systems Biology are working with AB Sciex to develop a new mass spec workflow that could significantly accelerate quantitative metabolomics analyses.
The new workflow, which is being developed on AB Sciex's QTRAP 5500 instrument, could reduce the time needed to conduct quantitative analysis of metabolites by up to ten-fold, Uwe Sauer, professor of systems biology at the ETH Zurich Institute of Molecular Systems Biology and leader of the research team, told ProteoMonitor.
As in proteomics, upstream chromatography is key to reducing the complexity of metabolomic samples prior to mass spec analysis. It can be a time-consuming process, however, limiting the number of samples that can be run, particularly given that metabolomic studies often deal with on the order of 1,000 different chemical species.
"We have quite often a lot more samples than we have measurement time," Sauer said. "If each method that you apply takes an hour, and to cover more than one set of metabolites you have to run three or four or even five methods in parallel, for each sample it's five times an hour. And when you do dynamic analysis [looking at] where metabolites are changing, they're changing on the second scale, so the community needs methods that operate on the time scale where you can measure samples that were taken every few seconds."
To get around this issue, Sauer and the ETH Zurich researchers are using the QTRAP 5500 to develop multiple-reaction monitoring assays for various classes of metabolites, eliminating the need for extensive chromatography beforehand. The instrument, which AB Sciex released in 2008, combines a triple quad for quantitation with a linear-ion-trap-based qualitative mode, allowing Sauer to build the assays using complex, unknown samples as opposed to sets of known standards.
"The traditional approach has been to start with a bunch of standards, develop MRM assays to those, and then look for those relatively smaller number of analytes in samples," Dominic Gostick, director of the biomarker mass spectrometry at AB Sciex, told ProteoMonitor. "[Sauer] is really starting with unknowns and targeting a lot more compounds simultaneously than other groups have."
"It's not necessary to use extensive chromatographic gradients on the front," Sauer said. "Because of the sampling rates and the mass accuracy of the instrument that's actually – if you really push it to the limit – not necessary anymore."
The 5500's combination of qualitative and quantitative workflows is key to the process, Gostick said, as it allows researchers to get full-scan mass spectrometry data for a sample using the ion-trap function and then develop MRM assays from that data for the triple quadrupole.
Such qual-quan combinations have been points of emphasis for AB Sciex. Upon the release of the 5500 two years ago, Andy Boorn, currently chief operating officer of AB Sciex and then president of MDS Analytical Technologies – which partnered with AB Sciex on the machine – highlighted the instrument's qual-quan capabilities, telling ProteoMonitor it would allow researchers to move from "sensitive, very specific triple quadrupole scan mode to highly sensitive full scan ion trap mode in less than 1 millisecond" (PM 10/16/2008).
At this year's American Society for Mass Spectrometry annual meeting the company released its new TripleTOF 5600 instrument, which Boorn said also combined qualitative and quantitative workflows, allowing researchers to "do the equivalent of multiple-reaction monitoring experiments [at] high sensitivity [and] high speed at the same time that you're acquiring full mass spectra, the whole mass spectrum, every peak from that sample at high resolution" (PM 05/28/2010).
"We're absolutely passionate about the idea of qual and quan on a single platform," Gostick said. "[Sauer's approach] is more targeted, more quantitative, but covering a broader number of analytes than some of the really targeted approaches that some of the other labs have used. It bridges this middle space. That, I think, is what is appealing here."
The researchers began work on the method roughly half a year ago. Sauer expects to have a usable version of the workflow completed and available to the metabolomics community in the next six months to a year.
"The key limitation now is when you shorten these gradients, when you shorten the runtime on the chromatography, how do you get the optimal instrument settings to still get information on separate compounds that co-elute almost at the same time," he said. "We're trying to get the last bit out of what the 5500 can do and possibly learn what the key issues are going to be for the next generation of instruments if you want to push that further."
His team is also considering building a database to contain the MRM assays it develops – similar to the MRM Atlas that Sauer's ETH Zurich colleague Ruedi Aebersold has developed for proteomics research.
"All the particular settings, plus whatever specifications on what the measurements will be, will be publicly available," he said.
Sauer envisions the method as an efficient first round of examining a sample, "perhaps with some subtraction of resolution, but still with quite good resolution and quantification, but at a tenth of the time."
"That would make a huge difference, and it would allow us to find out much faster what are the time phases or samples that we have to invest more time in," he said. "The real limitation quite often is that not every sample is equally important and not every metabolite in a sample is equally important."