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2012 Sees Rise of Targeted Proteomics and DIA-MS, Continued Clinical Push by Big Industry Players

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You might say 2012 was the year that proteomics shed its Rodney Dangerfield routine and began to get some respect.

The field has always had its proponents, of course. But there's been no shortage of pessimists either, particularly as its early, perhaps overhyped, promise faded, and a decade of research passed with relatively little to show in terms of clinical impact.

Pushing proteomics into the clinic continues to prove a challenge, as the 2012 struggles of protein diagnostic firms like Vermillion and BG Medicine demonstrated. However, large mass spec players like AB Sciex, Agilent, Bruker, and Thermo Fisher Scientific as well as established diagnostic outfits like Quest Diagnostics continue to invest in clinical proteomics efforts. And a number of smaller companies including Integrated Diagnostics, Sera Prognostics, and Caprion Proteomics are prepping new protein biomarker tests for launch in 2013.

Beyond that, several significant funding raises by proteomics firms as well as a major pharma acquisition suggest that the field, while certainly not without its difficulties, has over the last year achieved a firmer footing.

Proteomics on Target

One highlight for 2012 was the rapid adoption of targeted proteomics methods — a trend underscored by Nature Methods' decision to name the technique "Method of the Year."

"Interest in applying targeted mass spectrometry technology on a broader scale has been rapidly escalating in the past several years, and new methods, tools, resources and next-generation approaches are helping to bring the technology to a wider community of researchers," the authors wrote. And, indeed, 2012 saw significant advances in targeted proteomics research.

For instance, in November, researchers from Agilent and SISCAPA Assay Technologies published on a multiple-reaction monitoring mass spec-based peptide quantitation workflow with a sample cycle time as short as seven seconds (PM 11/16/2012). The technique, which achieved these cycle times by replacing traditional liquid chromatography with Agilent's RapidFire solid phase extraction system, enables a dramatic increase in the throughput of targeted protein assays — a key limitation in validating and implementing protein biomarkers..

As Leigh Anderson, CEO of SISCAPA Assay Technologies and author on the paper, told ProteoMonitor in an interview following the release of the study, while the work is not "the final word," it "does at least put a stake in the ground with respect to the kind of throughput that could ultimately be obtained in clinical [proteomic] assays."

Thermo Fisher Scientific likewise continued its targeted proteomics push, with the company's Biomarkers Research Initiatives in Mass Spectrometry Center leading a multi-facility study examining the inter-lab reproducibility of selected-reaction monitoring assays applied to clinical samples treated with upfront protein enrichment techniques (PM 6/8/2012).

The results of the effort, George Mason University researcher and study participant Emanuel Petricoin told ProteoMonitor, suggest that while considerable testing and optimization remains to be done, "you can perhaps start to see the potential for how clinical mass spectrometry in the proteomics arena can match what has been happening in the small-molecule field for decades now."

Hi-Res Revolution

Thermo Fisher was also at the forefront of one of the year's most notable targeted proteomics advances – the development of protein quantitation techniques using high-resolution mass spec instruments.

Targeted proteomics assays have traditionally been performed on triple quadrupole machines, which have offered higher sensitivity for such work than high-resolution machines like time-of-flights or Orbitraps. However, with the release of new Q-TOF and Q-Orbitrap instruments, and Thermo Fisher's Q Exactive, in particular, researchers can now achieve triple quad-level sensitivity on high-resolution instrumentation.

This is significant because, unlike triple-quads, high-resolution analyzers are able to collect data on a wide range of ions, giving the machines the potential for easier targeted assay development and better specificity.

Such high-resolution targeted assays were presented last year in papers published separately by the labs of University of Wisconsin-Madison researcher Josh Coon and Bruno Domon, head of the Luxembourg Clinical Proteomics Center, with both researchers using the Q Exactive for the work (PM 10/12/2012).

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 Coon's and Domon's research – titled parallel-reaction monitoring – use the upfront quadrupole of a Q-TOF or Q Exactive machine to isolate a target precursor ion, but then monitor 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.

While triple quads have remained the instrument of choice due to their perceived sensitivity advantages, both groups' studies 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.

Data Independence

Fundamentally, the PRM technique is a narrow application of another broader trend that gained significant ground within proteomics this year – data-independent acquisition mass spec (PM 6/8/2012).

Traditionally, shotgun proteomics experiments have used data-dependent acquisition wherein the mass spectrometer performed an initial scan of precursor ions entering the instrument and selected a sampling of those ions for fragmentation and generation of MS/MS spectra. Because instruments can't scan quickly enough to acquire all the precursors entering at a given moment, however, many ions – particularly low-abundance ions – are never selected for MS/MS fragmentation and so are not detected.

In DIA, on the other hand, the mass spec selects broad m/z windows and fragments all precursors in that window, allowing the machine to collect MS/MS spectra on all ions in a sample.

Enabled by the increasing scan speed of linear ion-trap instruments, the technique was introduced in a 2004 Nature Methods paper by Scripps Institute researcher John Yates III – who implemented it on a Thermo Electron LTQ instrument. Since then, DIA techniques have been employed by a number of proteomics researchers using a variety of different platforms.

But while the method has been around for close to a decade, 2012 was something of a watershed year for DIA, particularly with regard to its use in combining qualitative and quantitative proteomic analyses.

Notably, last year AB Sciex released its new TripleTOF 5600+ instrument featuring its Swath data-independent acquisition technique, perhaps the most fully developed such product currently offered by a major mass spec vendor.

As University of Washington researcher Michael MacCoss, whose lab's open-source Skyline software system has emerged as a major platform for DIA data analysis and method development, told ProteoMonitor, "what's different about what people are doing now is that they are saying, 'OK, for this peptide I can predict the mass; I can predict the fragment ion masses; can I now find it in the [DIA] data?'"

"What AB Sciex has done [with Swath] is that they were the first people to kind of push this [targeted] workflow," he said.

Other vendors are now focused on catching up, many of them in collaboration with MacCoss and his team's Skyline software tool, which currently supports DIA methods from companies including AB Sciex, Waters, Agilent, Thermo Fisher Scientific, and Bruker.

Cash Considerations

Beyond such technical strides, proteomics as a field also saw in 2012 some more strictly material signs of progress – perhaps most significant GlaxoSmithKline's May purchase of chemical proteomics firm Cellzome for $99 million (PM 5/18/2012).

Cellzome specialized in mass spec-based proteomic screens for measuring drug-protein interactions, a capability that "really applies across a wide spectrum of the drug discovery chevron," Bob Hertzberg, vice president of screening and platform profiling at GSK, told ProteoMonitor upon announcement of the deal. He cited among its potential uses compound selectivity profiling, mechanistic studies, and identifying potential off-target effects.

Several proteomics firms also managed significant fundraising rounds, with Integrated Diagnostics raising $10 million to support the 2013 launch of its lung cancer diagnostic (PM 2/24/2012), Applied Proteomics securing $22.5 million to support its protein biomarker discovery work (PM 2/10/2012), and Astute Medical completing a $40.4 million funding round (PM 6/8/2012) to support commercialization of protein biomarker tests including the company's Nephrocheck test for acute kidney injury, which launched in Europe in October (PM 10/19/2012).

The Downside

Of course, the year wasn't without its dark spots. Diagnostics firm Vermillion continued to disappoint investors, with sales of its OVA1 ovarian cancer test flat-lining and its credit agreement with Quest Diagnostics coming to an end (PM 10/19/2012). The company terminated CEO Gail Page in December, appointing board member Bruce Huebner as interim CEO while it searches for Page's permanent successor.

BG Medicine also struggled this year, with its stock price ending the year at $2.31 per share, down more than 50 percent from where it began 2012 and almost 75 percent off its 2012 high of $8.36. Although the company saw significant growth in sales of its BGM Galectin-3 heart failure test, it failed to achieve the revenue benchmarks required to draw down an additional $5 million under its loan agreement with General Electric, and withdrew its submission to the US Food and Drug Administration seeking 510(k) clearance for its CardioScore biomarker test for cardiovascular risk, saying it would not be able to respond in full to a request from FDA to confirm certain data from its validation study for the test (PM 11/16/2012).

The company did report some encouraging end-of-year news, however, announcing in December that it had received an EU CE mark for the CardioScore test. It also announced this week that it had received a CE mark for an automated version of its Galectin-3 test produced in collaboration with French diagnostics firm BioMérieux.

Beyond the difficulties of these particular firms, there continued to be concerns within the field more generally regarding current approaches to clinical proteomics. At a discussion session at the American Society for Mass Spectrometry annual meeting in May, representatives from several major clinical reference labs offered gloomy takes on the potential of protein biomarker tests, with one participant, Russell Grant, strategic director and national director of mass spectrometry at Laboratory Corporation of America, going so far as to say that given validation costs and the intellectual property landscape, he didn't currently "see a path" for new protein biomarkers to make their way into the clinic (PM 5/25/2012).

Clinical Push Continues

Yet, despite such bleak prognostications, a number of large vendors and diagnostics firms continued in 2012 to put their weight behind clinical mass spec and proteomics efforts.

For instance, Quest Diagnostics last year launched a mass spec-based thyroglobulin assay intended to aid in monitoring recurrence of thyroid cancer following surgery (PM 6/22/2012). It was the third mass spec-based protein test Quest has launched, following its angiotensin assay for plasma renin activity and an assay for insulin-like growth factor 1.

Agilent, meanwhile, announced in January that it was partnering with Integrated Diagnostics to develop mass spec workflows for protein biomarker diagnostics, with the two companies collaborating to improve existing MRM-MS techniques with the aim of moving Integrated’s proteomic tests for lung cancer and Alzheimer’s disease into the clinic (PM 1/13/2012).

Also in January, Agilent said that it had registered Infinity Series 1200 liquid chromatography systems and 6000 Series mass spectrometry systems as Class I medical devices with the US Food and Drug Administration, clearing the way for their use in clinical settings (PM 1/20/2012).

AB Sciex similarly took regulatory steps toward the clinic, achieving ISO 13485 certification for its liquid chromatography and mass spectrometry manufacturing facility in Singapore and R&D center in Toronto, a move president Rainer Blair called "an important milestone… along the path to the [European] CE [in vitro diagnostics] mark and subsequently the [US Food and Drug Administration] 510(k) approval for the mass spec in clinical diagnostics," during a company press conference at Pittcon in March (PM 3/16/2012).

And Thermo Fisher in October inked a collaboration deal with German diagnostics firm Immundiagnostik for development of mass spec-based assays for protein and peptide quantitation (PM 10/26/2012).

The deal will focus on development and commercialization of new LC-MS/MS assays to diagnose cardiovascular disease and bone metabolism disorders and offers Thermo Fisher a venue to refine and demonstrate its clinical mass spec workflows, in particular its Mass Spectrometric Immunoassay, or MSIA, technology.

Traditionally specializing in immunoassays, Immundiagnostik expects that mass spec's higher specificity and the potential cost savings enabled by its multiplexing ability will drive the growth of clinical interest in the technique, company spokesperson Susanne Kuhlendahl told ProteoMonitor upon announcement of the deal.

The company is "convinced that [mass spec] technology will more and more replace immunoassays in laboratory diagnostics," she said.

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