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MSACL 2015 Sees Continued Focus on Clinical Proteomics' Calibration, Standardization Challenges

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SAN DIEGO (GenomeWeb) – Mass spec might be the stated focus of The Mass Spectrometry Applications to the Clinical Laboratory annual meeting, but at the conference, held this week in San Diego, instrumentation itself was something of an afterthought. 

Rather, many of the gathering's presentations – at least where proteomics was concerned – centered around processes and workflows leading up to actual mass spec analysis, as researchers continue to refine assays for clinical deployment.

One example of the complexities involved in developing mass spec-based protein assays suitable for clinical use was provided by Laboratory Corporation of America researcher Christopher Shuford, who presented on his group's efforts to develop a mass spec assay for monitoring thyroglobulin levels in thyroid cancer patients.

Traditionally, such thyroglobulin testing has been done using immunoassays; however, patient auto-antibodies can make such tests ineffective. This has lead clinicians to pursue mass spec as alternative approach and, in the last several years, companies including Quest Diagnostics, Mayo Medical Diagnostics, and ARUP Laboratories have launched mass spec-based thyroglobulin tests.

The LabCorp presentation demonstrated, though, the many intricacies involved in building clinical mass spec assays. In particular, it focused on the challenge of building an appropriate calibrator for the test. The researchers explored multiple matrices – including sera from thyroidectomy patients in remission, animal sera including from chicken and pig, and an artificial matrix consisting of human serum albumin diluted into PBS – combined with multiple sources of thyroglobulin, including BCR Certified Reference Material, Beckman Access Thyroglobulin Assay Calibrators from Beckman Coulter, human-derived thyroglobulin from Sigma-Aldrich, and native thyroglobulin in serum.

As Shuford showed, performance of the calibrators was dependent on factors including storage time and was subject to various matrix effects that in some cases decreased analytical sensitivity.

As at last year's meeting, the challenges of trypsin digestion were also a topic of interest, with Kevin Meyer, executive vice president and CSO of proteomics sample prep firm Perfinity Biosciences, presenting on his company's research into the vagaries of the digestion process.

Among their less anticipated findings, Meyer said, was the fact that the choice of buffer results in significantly different levels of trypsin activity.

The general assumption, he said, has been that trypsin was little affected by the reagents used for preparing target proteins for digestion. But, he noted, "trypsin is a protein, so whatever you do to denature your targets, that will affect your trypsin."

Meyer and his colleagues also found dramatic differences in the times required for different proteins to digest completely, a fact that he noted could have implications for what proteins researchers will see in their shotgun proteomics experiments.

Irene van den Broek, a researcher from Leiden University Medical Center, likewise focused on issues of digestion, presenting findings from work that her group published several months ago on sources of error and imprecision in MRM mass spec workflows measuring the serum apolipoproteins Apo A-1 and Apo B.

One of the key questions van den Broek and her colleagues considered was what kind of standards to use and where in the process to use them. She noted in her presentation that while stable isotope-labeled peptides were sufficient to achieve high reproducibility in their measurements, they did not account for differences in digestion efficiency that could lead to assay inaccuracy. One possibility, she said, was external calibration using native, value-assigned sera. 

Stephen Hunsucker, senior director of laboratory operations at Integrated Diagnostics, presented on that company's approach to controlling variability in its Xpresys Lung test, the first multi-analyte multiple-reaction monitoring mass spec proteomics assay to make it into the clinic.

The Indi test uses the relatively unique approach of controlling for assay variation through both traditional stable isotope-labeled peptide standards and a set of endogenous protein standards that allows for the control of pre-analytical variation caused by for instance differences at sample collection sites.

Of the 11 proteins the Xpresys test measures, only five actually have diagnostic value. The remaining six are used as normalizers, which serve to control for systematic variation throughout the pre-analytical and analytical portions of the assay.

Combining use of these six endogenous normalizers with the addition of SIL peptide standards post-digestion to control for variation in desalting and mass spec analysis, the Indi researchers achieved a median CV of all proteins measured of 9.3 percent, compared to 13.3 percent using SIL peptides alone.

Of course, some presentations did focus on the actual mass spec portion of the workflow. For instance, Luxembourg Clinical Proteomics Center head Bruno Domon provided an update on his work developing high-resolution accurate mass instruments for targeted protein analysis.

In a triple quad-based MRM assay, the first quadrupole isolates a target precursor ion, which is then fragmented in the second quadrupole, after which a set of pre-selected product ions are detected in the third quadrupole. By contrast, a high-resolution approach – typically termed parallel-reaction monitoring, or PRM – uses 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 using its time-of-flight or Orbitrap analyzer.

In theory, this offers several advantages compared to conventional SRM. For example, because the technique monitors all product ions instead of just a pre-selected few, researchers don't have to determine upfront what the best transitions to monitor will be, significantly reducing assay development time.

Additionally, the larger number of product ions monitored via PRM should 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.

This week, Domon presented on a new acquisition method he called internal standard triggered-parallel-reaction monitoring (IS-PRM), which uses internal standards to guide the adjustment of acquisition parameters like resolution and fill time to optimize measurement of target analytes.

And, on the microbiology front, a number of presentations suggested the field is looking beyond MALDI mass spec, which has to date dominated proteomic-based microbial ID.

For instance, Tiphaine Cecchini, a researcher from BioMérieux – with Bruker one of the two main players in MALDI-based microbial ID – presented work on assessing antibiotic resistance in Acinetobacter baumannii using selected-reaction monitoring. And University of California, San Diego researcher Alec Saitman presented on Abbott Diagnostics' Iridica platform, an ESI-TOF mass spec system that the company said can produce bacterial IDs directly from blood specimens in less than eight hours.

Additionally, Waters' rapid evaporative ionization mass spectrometry (REIMS) technology, which the company acquired last year through its acquisition of MediMass, was the topic of considerable conversation, particularly given its usefulness in identifying microorganisms via lipidomic profiles, which a number of researchers considered a potentially promising approach to clinical microbial ID.

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