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U-Vic Proteomics Centre Pushing Multiplexed MRM-MS for Protein Biomarker Discovery

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Researchers at the University of Victoria, Genome British Columbia Proteomics Centre have developed a multiplexed multiple-reaction monitoring mass spec assay capable of quantifying 142 proteins in undepleted and non-enriched human plasma in a single run.

According to Christoph Borchers, director of the center, the assay, which was described in a paper published last month in Biochimica et Biophysica Acta, is among the most highly multiplexed MRM-MS assays developed to date. In total, the center currently has MRM assays to roughly 500 proteins, including 1,000 peptide standards, and aims by the end of the year to have 3,000 to 4,000 peptide standards for use in protein quantitation.

Ultimately, Borchers told ProteoMonitor, the researchers hope in collaboration with others to develop MRM assays along with the necessary peptide standards for every protein in the human proteome, – around 20,000 assays. At a development cost of roughly $1,000 per assay, such an effort would run around $20 million, he said, noting that, while not an inexpensive undertaking, the price tag would be considerably lower than, for instance, the more than $1 billion spent on the Human Genome Project.

"I see it as an international effort," Borchers said, citing the Human Proteome Project as a vehicle through which such work might be coordinated. "And certainly there is a huge commercial opportunity as well if companies like Sigma-[Aldrich] or Thermo [Fisher Scientific] were to try to bring [such an assay collection] to market."

Borchers and the Genome BC Proteomics Centre researchers offer their MRM assays through the company MRM Proteomics, a firm spun out from the center to commercialize its protein quantification technologies. Currently, the company, of which Borchers is the chief scientific officer, offers a variety of MRM-based products, including its Human Discovery MRM Assay, which quantifies levels of 104 proteins linked to various diseases; its Human Cardiovascular MRM Assay, which quantifies 84 proteins involved in cardiovascular disease; and its Mouse Discovery Assay, which quantifies 34 disease-related proteins. The company also offers custom MRM assay development services.

Borchers noted that while MRM mass spec has received the most attention as a potential technique for biomarker verification and validation, he and his colleagues "want to use it for [biomarker] discovery."

In this endeavor they are following the not entirely successful pathway of defunct proteomics firm NextGen Sciences, which over the last several years had worked on developing MRM-based biomarker discovery panels in hopes of competing with similar immunoassay-based products offered by Myriad RBM – the leading player in the protein biomarker discovery panel space.

In 2011 NextGen launched its first such product – plasmadiscover41, a panel comprising assays to 41 plasma proteins thought to be potential biomarkers for breast, prostate, and lung cancer. It followed this with a number of additional panels, including an expanded human plasma panel and cerebrospinal fluid panels for human and rat, but it was ultimately unable to generate sufficient revenues from this business, and in January of this year the company's protein quantitation assets were purchased by start-up biotech firm Molecular Metabolism (PM 5/31/2013).

Compared to immunoassays, MRM mass spec offers a number of advantages, including reduced assay development times and costs, as well as a significant jump in multiplexing capabilities. One factor that continues to limit MRM mass spec, however, is the technique's relatively low sensitivity.

For instance, the 142 proteins quantified in the MRM method recently developed by Borchers and his team ranged in abundance from 31 mg/mL to 44 ng/mL. Though useful markers exist in this range, many potential biomarkers – proteins leaking from tumor tissue into the bloodstream, for instance – are present at low pg/mL concentrations.

The Genome BC researchers are currently developing MRM assays using pre-fractionation that can push into that lower range, Borchers said. He noted that they could achieve even higher sensitivity using immunoenrichment-based methods like SISCAPA; however, he said, if possible they hoped to avoid such approaches due to their added expense and complexity.

One encouraging sign on the sensitivity front, Borchers said, is the recent release of several new triple quadrupole systems that could offer improved performance compared to existing models. At the American Society of Mass Spectrometry annual meeting last month, Bruker and Thermo Fisher both launched new triple quads, with Bruker launching its EVOQ Elite ER instrument and Thermo Fisher its TSQ Endura and TSQ Quantiva machines (PM 6/14/2013).

For the BBA study, Borchers and his team used Agilent's 6490 instrument, which, he told ProteoMonitor last year, he had found offered the best sensitivity among machines then available (PM 10/12/2012). This week, however, he suggested that the new Thermo Fisher and Bruker instruments, along with AB Sciex's 6500 line, could provide improvements along this line.

"I have heard that the newest generation [of triple quads], particular the Thermo, Bruker, and AB Sciex instruments are more sensitive than the current [machines]," he said. "Of course, this puts us under pressure to buy these new instruments ... but I'm very happy that the companies are really pushing the instrumentation for more sensitivity."

A smaller 120-protein panel drawn from the 142-protein assay presented in the BBA study is currently being used by University of Toronto researcher Ahmed El-Sohemy for a large-scale nutrigenomics study investigating protein biomarkers of dietary exposure. That project is currently analyzing 438 human plasma samples using the panel with plans to analyze some 3,600 samples, Borchers said.

The Genome BC Centre researchers are also in discussions with Montreal's Jewish General Hospital on a collaboration, under which they would analyze the plasma proteome of every cancer patient admitted to the hospital, Borchers said.

For these analyses, the researchers would likely use MRM assays as well as shotgun-style mass spec, he said, noting that he was particularly intrigued by Thermo Fisher's new Orbitrap Fusion instrument. It "looks like it will be an ideal instrument for shotgun proteomics," he said.

Borchers added that at the Human Proteome Organization's 2013 annual meeting in September, MRM Proteomics plans to launch an MRM mass spec assay kit designed to let researchers benchmark their instrument's performance.

The kit will consist of assays to 14 proteins covering three orders of magnitude and will contain peptide standards as well as protocols "to check out the performance of your instrumentation ... is it at optimal performance, or is it dirty, or is the LC working correctly and so on," Borchers said. The kit will be co-marketed with Agilent.

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