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Studies Make Case for High-Res MS as Triple Quad Alternative, Though Questions and Challenges Loom

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Last month, a research team led by Bruno Domon, head of the Luxembourg Clinical Proteomics Center, published a paper in Molecular & Cellular Proteomics demonstrating the use of Thermo Fisher Scientific's Q Exactive mass spectrometer for targeted protein quantitation.

The study, which follows a similar paper published in MCP in August by University of Wisconsin-Madison researcher Josh Coon (PM 8/10/2012), offers additional evidence for the potential of high-resolution instruments as an alternative to triple quadrupoles for selected-reaction monitoring-style quantitation.

The triple quadrupole is currently the standard instrument for targeted protein quantitation, and, as such, is the primary platform for much pharmaceutical and clinical proteomics work.

High-resolution quadrupole-based devices like the Q Exactive or the various Q-TOF machines from vendors including Waters, AB Sciex, Agilent, and Bruker, however, offer potential advantages over triple quadrupoles. Specifically, because their high-resolution Orbitrap or time-of-flight analyzers are able to collect data on a wide range of ions, the machines have the potential for easier assay development and better specificity.

In a triple quad-based SRM assay, the first quadrupole isolates a target precursor ion, which is then fragmented in the second quadrupole, after which a set of preselected product ions are detected in the third quadrupole. By contrast, the high-resolution approaches detailed in the Coon and Domon papers – titled parallel-reaction monitoring – use the upfront quadrupole of a Q-TOF or Q Exactive machine to isolate a target precursor ion, but then monitors not just a few, but all of the resulting product ions.

Because of this, researchers don't have to determine upfront what the best transitions to monitor will be, significantly reducing assay development time.

The larger number of product ions monitored via PRM should also improve the specificity of the analysis, since more transitions will be available to confirm a peptide ID. This might also reduce the effects of co-isolating background peptides.

Nonetheless, despite these potential benefits, triple quads have remained the instrument of choice for quantitation due in large part to their perceived advantage in sensitivity.

Both the Coon and Domon studies, however, found that in comparisons of the Q Exactive with, respectively, Thermo Scientific's Quantum Discovery Max and TSQ Vantage triple quadrupoles, the high-resolution instrument offered on average equivalent or better sensitivity.

"Overall, if you compare the data, the specific numbers like sensitivity, they are comparable," Domon told ProteoMonitor.

As Coon told ProteoMonitor upon release of his group's study, while triple quadrupole detectors offer more raw sensitivity than a high-resolution analyzer like an Orbitrap, the higher specificity of the latter machine compensates for this.

"The Orbitrap isn't as sensitive, but it can [better] discriminate the ions of your target from the background, so what you lose in sensitivity you gain in selectivity, and it turns out you break even," he said.

Given its newness, high-resolution-based targeted quantitation techniques like PRM have thus far been confined mostly to cutting-edge academic mass spec labs. However, Domon said, the technique has potential to move into the clinical and industrial spaces where triple quad-based SRM work has traditionally dominated.

He noted, though, that it will take time for the technique to be adopted. "I think it has great potential, but it is a different way of doing quantitation, and it will take some time to get it truly accepted by the clinical community and the analytical chemists."

PRM-based quantitation "sounds potentially very attractive, and it makes sense to look at this alongside [triple quadrupole- and MALDI-TOF-based quantitation]," Leigh Anderson, CEO of SISCAPA Assay Technologies and a leading protein quantitation researcher told ProteoMonitor in an email. However, he agreed with Domon that "it will take some time to get to the comfort level of [triple quads] for most large-scale users."

Domon suggested that rather than replace triple quadropoles for existing assays, high-resolution machines might make inroads by researchers employing them in newly developed targeted quantitation assays.

"If a company already has an assay established and they have 20 triple quads doing it, they are not going to switch," he said. "I think what might happen is that pharmas and biotech will look now at this new technology and as new assays come along they might develop those on these [high-resolution] platforms."

"The triple quad has been really established and used in the clinic for small-molecule analysis for two decades," Domon added. "So it is a mature technology and has been well tested and you have protocols. [High-resolution quantitation] is a new technology, so it will need a little more work to actually get the protocols and all that it needs to make it a clinical device."

He said he was unaware of any industry parties currently using the Q Exactive for an established targeted protein quantitation protocol.

However, pharmaceutical firm Sanofi is using the instrument for selected-reaction monitoring assays as part of an Alzheimer's protein biomarker development effort, suggesting that high-resolution instruments are making some inroads as industry tools for targeted protein quantitation. Run out of the company's Exploratory Unit at Sanofi R&D, the project is focused on validation of a set of previously identified cerebrospinal fluid Alzheimer's markers in a new set of CSF samples.

Still, issues remain regarding the new technique – price, for instance. High-resolution machines are typically more expensive than triple quads, a potential barrier noted by Ian Pike, chief operational officer of Proteome Sciences, which offers a variety of SRM assays and targeted proteomics services.

"Everything I have seen from the Domon lab suggests [the Q Exactive] is an interesting alternative for SRM," he told ProteoMonitor. However, he added, "our current thinking is that in terms of price point the [Thermo Scientific] TSQ Vantage [triple quad] still offers the best option for targeted measurements right now."

Christoph Borchers, director of the University of Victoria – Genome British Columbia Proteomics Centre and founder and chief scientific officer of targeted proteomics firm MRM Proteomics, also cited the cost barrier, noting that the Q Exactive costs roughly $100,000 more than a high-end triple quad.

Borchers also expressed skepticism about the Coon and Domon findings that the Q Exactive could compete with high-end triple quads on sensitivity.

"I have my doubts that it will be as sensitive as triple quadrupole-based MRM," he told ProteoMonitor. "Triple quadrupoles are established in the field. They have been used for decades."

Noting that both Coon and Domon have relationships with Thermo Fisher – indeed, researchers from the company co-authored the Domon MCP paper – Borchers said he would like to see their results confirmed by additional teams.

He added that the triple quadrupoles used for comparison in the two MCP studies were perhaps not the most sensitive instruments, saying that in his research he had found Agilent's 6490 triple quad to offer the best sensitivity among currently available machines.

Borchers has a relationship with Agilent through MRM Proteomics as the two companies last month signed a co-marketing agreement.

PRM "is an interesting technology, but I have my sincere doubts that it can reach the sensitivity of a high-end triple quadrupole," he said.

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