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Targeted Mass Spec Slowly but Steadily Moving Toward Broader Commercial, Clinical Adoption

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While targeted mass spec techniques like multiple-reaction monitoring continue to wrestle with issues of sample preparation, throughput, and sensitivity, recent publications and presentations suggest that the technology has gained a foothold in both industry and academia as a research tool.

In particular, last week's 11th annual meeting of the Human Proteome Organization in Boston saw a number of scientists highlight targeted mass spec workflows for a variety of applications including protein biomarker verification and validation as well as pharmaceutical research.

Presenting work using immunoaffinity mass spec for preclinical pharmacokinetic/pharmacodynamic analysis of antibody-based drugs, Pfizer associate research fellow Hendrik Neubert observed that he has seen "an increased desire to use mass spectrometry, and specifically immunoaffinity mass spectrometry," for measuring target and therapeutic levels to support development of PK/PD models.

This increased interest in mass spec-based quantitation has "been driven by the need for very high specificity," Neubert said, adding that "we're often being presented in my area with questions where [conventional] immunoassays" can't provide this needed specificity.

He provided an overview of his group's immunoaffinity mass spec workflow, which is fully automated on a liquid handling platform and uses column-based antibody enrichment to capture target peptides and proteins for LC-MS/MS quantitation. Consisting of a high-flow antibody enrichment step followed by a short reverse-phase nanoflow chromatography stage, the platform enables assay runtimes of less than ten minutes per sample.

"The quality and the cost [of immunoaffinity mass spec] is becoming compatible with routine protein quantitation in translational research and the clinical environment," Neubert said. "So I expect at least in my area [of PK/PD modeling], and maybe in pharma in general, that there will be implementation of this kind of technology with increasing precedence."

Another industry player — French biotech firm BioMérieux — also reported on its use of targeted mass spec, with researcher Geneviève Choquet-Kastylevsky presenting on the company's use of MRM-MS to evaluate 57 candidate proteins as biomarkers for colorectal cancer.

Initially, Choquet-Kastylevsky said, BioMérieux had planned to develop ELISAs for the evaluation process but soon realized that developing nearly 60 ELISAs for verification wasn't feasible. The company then turned to mass spec, establishing an MRM-MS workflow employing normal-flow LC, which it used to quantify the candidate markers in two cohorts, one with 116 cases and 121 controls and the other with 115 cases and 222 controls.

Calling the project a proof of concept that confirmed "it was possible [to use MRM-MS] to quantify things like tumor markers in serum from a large number of patients," Choquet-Kastylevsky said the mass spec workflow offers "a technology platform which will really allow us to assess the performance of new biomarkers."

The effort "showed us that [the MRM-MS] approach was feasible," she added. "We think it is possible to achieve the development of such assays in humans."

Amanda Paulovich, a researcher at Seattle's Fred Hutchinson Cancer Research Center, likewise presented on MRM-MS, describing a collaboration between her group, Broad Institute researcher Steve Carr, and Seoul National University researcher Youngsoo Kim in which the teams developed four multiplexed assays consisting of 150 MRM-MS assays each, which she said they are using to measure expression of 300 proteins across 30 "widely used and extensively genomically characterized breast cancer cell lines."

Presenting results generated via analysis of twelve of those cell lines, Paulovich noted the high level of correlation between data generated in her lab and Kim's, observing that the assays demonstrated "not only an unprecedented level of multiplexing with this [MRM-MS] technology, but also that the assays were portable… halfway around the globe and across a language and cultural barrier."

MRM-MS, she said, offers assays that are quantitative, high-throughput, readily multiplexed, and reproducible.

Paulovich noted, however, that hurdles still remain for MRM-MS and immunoaffinity mass spec techniques like those described by Neubert. The technology falls short, she said, in that "we don't have analytically validated reagents available for the vast majority of proteins and the assays are not easy to generate de novo in most end-user laboratories in terms of time, money, or required expertise."

Echoing comments made by Swiss Federal Institute of Technology Zurich researcher Ruedi Aebersold during his HUPO talk last week (PM 9/14/2012), Paulovich suggested that to achieve its potential, MRM-MS must, like genomics, become a tool that can be easily used by "translational scientists that don't have specialized training in proteomics."

This goal is "eminently achievable," Paulovich said, adding that she believed "the proof of concept is here."

She acknowledged, however, that broader implementation will likely prove a challenge, noting that "there is a huge potential market, but it doesn't exist yet. So the commercial sector is afraid to invest in this on a big level and the academic sector doesn't have the money or the ability to do product development."

Despite this issue, however, targeted mass spec continues to move slowly but steadily into the commercial and clinical spaces. Last week, for instance, Agilent, which has been one of the primary industry players in the development of MRM-MS workflows, said it had signed a co-marketing agreement with MRM Proteomics, a company spun out of the University of Victoria-Genome British Columbia Proteomics Centre to commercialize targeted proteomic techniques developed there.

Under the agreement, Agilent will market MRM-MS kits developed by MRM Proteomics, Steve Fisher, Agilent metabolomics and proteomics marketing manager, told ProteoMonitor.

"They are in the process of developing kits that will help people doing quantitative proteomics, and we'll be marketing those kits," he said. "This will let us promote what they are doing and will help us in our business plan to promote triple-quad [quantitation] for protein measurements."

The pact is only the latest in a series of efforts by Agilent to develop MRM-MS as a tool for broad research and clinical applications. In January, the company announced a collaboration with biotech firm Integrated Diagnostics on improving existing MRM-MS techniques with the aim of commercializing InDi's lung cancer test (PM 1/13/2012). InDi aims to bring that diagnostic to market as a mass spec-based laboratory-developed test early next year.

Agilent has also partnered with Leigh Anderson, one of the inventors of the immunoaffinity-MS method SISCAPA and the founder and CEO of SISCAPA Assay Technologies, which has collaborated with Neubert and Pfizer on some of their immunoaffinity-MS work. The company and Anderson have been working for several years on automated SISCAPA workflows with the aim of achieving the high reproducibility and throughput necessary for clinical applications (PM 2/11/2011).

In an interview with ProteoMonitor this week, Anderson said that he and Agilent continue to work on methods to improve the speed and reproducibility of the workflow's trypsin digestion step, but noted that the SISCAPA workflow, which uses antibodies to enrich for peptides of interest prior to mass spec analysis, can measure medium- to high-abundance targets at a rate as fast as seven seconds per sample using Agilent's RapidFire system.

For low-abundance markers, which require more extensive chromatography, the system is capable of cycle times in the three- to five-minute range, Anderson said.

Fisher noted that sample prep remains the most challenging part of Agilent's efforts to develop automated, high-throughput workflows for both SISCAPA and standard MRM-MS.

He said, though, that the company "is very close to having all the bugs in the workflow worked out," adding that "we have some collaboration efforts going on right now, and I would expect we will definitely have published results within the next year."

Fisher also said Agilent hoped to begin marketing efforts for the first MRM Proteomics kits by the end of the calendar year.

Vendors beyond Agilent are working to improve the performance and accessibility of targeted mass spec workflows, as well. Thermo Fisher Scientific, for instance, recently led an effort that brought together several leading proteomics teams to examine the inter-lab reproducibility of MRM-MS assays applied to clinical samples treated with upfront protein enrichment techniques (PM 6/8/2012).

Bruker, which has traditionally focused its targeted proteomics efforts around its MALDI-MS instruments (PM 1/30/2012), this month launched its first LC-triple quadrupole instruments, giving the company new machines capable of running MRM-MS workflows (PM 9/7/2012).

And outside the tools space, some protein diagnostics firms are also moving toward targeted mass spec as platforms for their tests. In addition to InDi's aforementioned plans to launch a mass spec-based lung cancer test next year, biotech firm Sera Prognostics plans to launch its preterm birth prognostic next year on a mass spec platform (PM 11/18/2011).

And, in June, Quest Diagnostics began offering its new mass spec assay for thyroglobulin, intended as a test to aid in monitoring recurrence of thyroid cancer following surgery (PM 6/22/2012).

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