Data presented at the Human Proteome Organization's 10th annual meeting last week indicates that Thermo Fisher Scientific's new Q Exactive mass spectrometer approaches the quantitative capabilities of a high-end triple-quadrupole machine — supporting claims that company officials made upon launching the system at the American Society for Mass Spectrometry annual meeting in June.
At ASMS, Ian Jardine, Thermo's vice president of global R&D, told ProteoMonitor that preliminary data showed the instrument could potentially approach the quantitative capabilities of a high-end triple-quad, but noted it would take several months to verify whether that was indeed the case (PM 06/10/2011).
Last week at HUPO, Bruno Domon, director of the Luxembourg Clinical Proteomics Center and an early Q Exactive user, presented research that backed up those initial findings. In experiments comparing protein quantitation on the Q Exactive to a triple-quadrupole, Domon's team found that the two platforms were roughly comparable and concluded that "there is a lot of potential" for the Q Exactive as a quantitative machine.
"Initially, this instrument was more thought of as in the line of a [typical] Orbitrap instrument – a qualitative, protein-identification instrument," Domon said during the presentation. "To our surprise it was very quantitative. It has a large dynamic range, and we can quantify with it almost as well as on a triple."
The Q Exactive is essentially a modified version of Thermo Fisher's Exactive benchtop Orbitrap instrument but with a quadrupole up front for MS/MS workflows. The device has been positioned primarily as a competitor to Q-TOF machines, but, as Domon's research suggests, it could also prove an alternative to triple quadrupoles for targeted protein and peptide quantitation work.
Domon conducted a series of studies comparing the platforms. In one set he used the Q Exactive's targeted select ion monitoring, or SIM, mode, which quantifies peptides based on precursor ions, to measure a preparation of eight peptide isotopologues mixed at different concentrations. In those studies, Domon said, the two platforms produced "very similar results," with the Q Exactive providing "large dynamic range and very good sensitivity with limits of detection in the low attamole range if we concentrate the amount of peptides which had been injected on the column."
To test the Q Exactive in biological samples, Domon's lab performed a similar comparison using the instrument's SIM mode to look at a series of proteins in urine that the researchers are investigating as biomarkers for blood cancer.
In that experiment, "we saw that the consistent limit of detection on the triple quad was around 100 attamoles," he said. "And with the Q Exactive some of [the peptide measurements] are better and some are not as good, but overall it looks like they are quite comparable."
The researchers also tested the Q Exactive's performance in MS/MS mode, measuring both the urine proteins as well as several potential lung cancer protein biomarkers in plasma. Domon said they found "very good correlation" between the results obtained on the two platforms.
He cautioned, however, that "we only have a very small number of studies" comparing the two systems and that "there is more work to be done to come to a final conclusion" as to the Q Exactive's usefulness as a triple-quad alternative.
He also noted that, while experimental design on the instrument is theoretically simpler than on a triple-quad due to the fact that the Q Exactive collects data on all fragment ions, removing the need to select particular transitions for analysis beforehand, getting optimal results can still involve considerable adjusting of parameters.
"We used the Q Exactive next to the triple-quad and sometimes it was better and sometimes it was not as good, and if it's not as good we can tweak this parameter and that parameter," he said. "So on the one hand, I would say it's easier than a triple quad – you don't have to do all the experimental design. On the other hand, I would say, if there are 20 parameters you can tweak to make [the performance] better, then I don't know."
According to Alexander Makarov, Thermo Fisher's director of global research for life sciences mass spectrometry and principal inventor of the Orbitrap mass analyzer, the Q Exactive's potential as a quantitative instrument stems from the high transmission, linearity, and dynamic range of the Orbitrap detector and its multiplexing ability. Because the instument can collect multiple precursor or fragment ions in its C-Trap and then send them on to the Orbitrap for analysis in parallel, the instrument can run at high resolution while maintaining high enough throughput to keep up with ultra-high-performance liquid chromatography systems.
"We are utilizing the speed of mass selection by the quadrupole mass filter," Makarov told ProteoMonitor. "That means that if we need just 10 milliseconds to fill the C-Trap for a particular ion, then we can use the remaining milliseconds for filling with other ions" like internal standards or additional targets.
"All of [these ions] can be added together and fragmented together and analyzed together without losing time, because we now can store ions in the C-Trap in parallel for detection in the Orbitrap analyzer," he said. "That means that we can decouple the speed of the Orbitrap from the speed of the analysis."
During his presentation Domon provided an example of the usefulness of the Q Exactive's resolving power, observing that by setting the machine at its maximum resolving power of 140,000 FWHM, the researchers were able to quantitate a peptide spiked into yeast that, at lower resolution, had been difficult to separate from background peaks. As he noted, this bump in resolution came at the expense of the instrument's duty cycle – the trade-off the multiplexing described by Makarov could mitigate.
"Because [with an Orbitrap] you are acquiring the full spectrum, you see all the fragments of each precursor ion, so you get all the transitions you should expect from this precursor," Makarov said. "Then, the multiplexing comes in because you have a high-resolution spectrum that is sparsely populated, so you can populate it more densely by adding more and more different precursors into the same spectrum and still not overlap the fragment peaks."
This raises the possibility, Jardine said, that the machine might be able to "do high-sensitivity, high-precision quantitation of peptides, but at even higher throughput than triple quads."
Additionally, Makarov said, the Q Exactive could make inroads in quantitation experiments that are measuring multiple transitions per precursor ion.
"Triple quadrupoles are unbeatable when you are looking at single transitions, with SRM [assays]" Makarov said. "And many applications can be done with conventional SRM, and there's no point to competing with the triple quadrupole in this area. But when you have multiple transitions for each precursor … the more transitions, the more benefit the Q Exactive could provide."
He estimated that applications with four to five or more transitions per precursor "could best use the Q Exactive, and that is where we expect it to take over."
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