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At MSACL 2014, Advances in MS-based Clinical Proteomics Draws Increased Scrutiny of Field's Methods

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SAN DIEGO – The Mass Spectrometry Applications to the Clinical Laboratory annual meeting arrived this week in San Diego with mass spec-based clinical proteomics having made significant strides in the last year.

In October, for instance, Integrated Diagnostics launched its Xpresys Lung test, the first multiplexed proteomic test to go to market using multiple-reaction monitoring mass spec on a triple quadrupole instrument.

In another milestone for the field, 2013 saw both Bruker's MALDI Biotyper and BioMérieux's Vitek MS proteomic microbiology systems receive US Food and Drug Administration 510(k) clearance, making them available for US clinical use.

This week's meeting suggested, however, that such successes will likely bring with them increased scrutiny, as mass spec-based proteomics moves out of the research realm and into the domain of clinical chemists. While much of the discussion at past MSACL meetings has centered around broad steps required for mass spec-based proteomics to demonstrate clinical workability, this year evinced a shift toward more narrowly focused concerns – an effort to work out the finer details of the method's reproducibility and portability.

One way of viewing this shift, said SISCAPA Assay Technologies CEO Leigh Anderson, is that "all of this original proteomics research has moved enough in the direction of [clinical chemistry] that [clinical chemists] are now asking questions that they care about, and not just saying 'OK guys, come back to me when this is really working.'"

And as mass spectrometrists at firms like Quest Diagnostics and Laboratory Corporation of America increasingly examine mass spec-based protein analysis from the point of view of a large reference laboratory, questions of obtaining accurate, reproducible results on time and cost scales appropriate to such laboratories are moving to the fore.

A small, specialized firm like Indi can build a business based on a relatively high-priced, low-throughput test, Anderson noted. Indeed, as the company told ProteoMonitor upon launch of the test, the Xpresys Lung costs several thousand dollars and takes around ten days to process.

A large reference laboratory, on the other hand, needs high volumes and low prices, "so it's a different set of challenges," he said.

And while Indi can focus single-mindedly on perfecting its proteomic workflow, a firm like LabCorp "has 99.9 percent of its volume in arenas [like small molecule analysis], and only a little bit of mass spec-based protein work," SAT board member Gus Salem told ProteoMonitor.

This is not to say, of course, that large reference labs are entirely new to mass spec-based protein analysis. Quest Diagnostics, for instance, has launched three mass spec protein tests: an angiotensin assay for plasma renin activity, an assay for insulin-like growth factor 1, and a thyroglobulin assay to aid in monitoring recurrence of thyroid cancer following surgery.

Among the various parts of the mass spec proteomic workflow, the reproducibility of trypsin digestion emerged as perhaps the most significant area of concern at this week's meeting, with researchers including Russell Grant, LabCorp's VP of research and development; Andy Hoofnagle, director of clinical mass spectrometry at the University of Washington; and Irene van den Broek, a researcher at Leiden University Medical Center, discussing this issue in their presentations.

Additionally, a number of other presenters were asked questions regarding their protocols for ensuring reproducible trypsin digestion, including Stephen Hunsucker, Indi's senior director of laboratory operations, who presented on the company's development and validation of the Xpresys Lung test. Indi is currently preparing a paper for publication detailing its analytical validation of the test, Hunsucker said.

Of course, one potential way around the concern about the reproducibility of trypsin digestion is to simply skip the digestion step and measure intact proteins. Arizona State University researcher Dobrin Nedelkov presented such a method for measuring IGF-1, showing data from a study in which he and his colleagues used immunoenrichment combined with MALDI mass spec to measure IGF-1 levels in 1,000 human plasma samples in a single day.

Nedelkov was formerly CEO of Intrinsic Bioprobes, an ASU spinout launched to commercialize the mass spectrometric immunoassay (MSIA) technology developed by his fellow ASU researcher Randall Nelson. Intrinsic Bioprobes was purchased by Thermo Fisher Scientific in 2011 and Nelson and Nedelkov continue to collaborate with the company on MSIA-based protein biomarker assays.

This work with Thermo Fisher typically uses MRM-MS workflows on triple quadrupole instruments. Nedelkov told ProteoMonitor this week, however, that in their internal work, the ASU researchers primarily use MALDI MS at the present time.

Most clinical proteomics work, including the majority of the targeted proteomic presentations at this week's MSACL meeting, has focused on MRM-MS workflows. MALDI's potential advantages in terms of throughput and ease of sample prep and operation, however, have led several researchers including Anderson, Stanford University researcher Mark Stolowitz, and University of Victoria researcher Christoph Borchers to explore the method for targeted protein quantitation.

Additionally, Nedelkov noted, with this year's FDA 510(k) clearance of the Bruker MALDI Biotyper and BioMérieux's Vitek MS systems, MALDI mass spec platforms have passed a key regulatory hurdle.

"It's already in the clinic for microbiology," he said. "It's been accepted. Now, the key is to find a killer app for protein diagnostics. All the elements are there. So the thing is, who now is going to package something into a kit that gets 510(k) clearance for mass spec detection?"

Nedelkov noted that one potential disadvantage for MALDI in terms of clinical acceptance is the lack of familiarity with such platforms in large reference labs, which have for years now used triple quadrupoles for small molecule work.

"They don't have a MALDI, so they'll need proof of concept and validation done somewhere else before they jump in," he said, adding that the trypsin digestion concerns on display at this week's meeting could also help provide an opening for the technology.

People "are scared of digestion, and I understand why," he said. "We don't like to digest things if we can detect them at the intact level."

Nedelkov noted that LC-MS systems could also potentially be used for clinical assays of intact proteins, citing in particular work on Thermo Fisher's Q Exactive instrument quantifying intact insulin.

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