MRM verification assays for candidate cardiac biomarkers

Proteomics researchers are rapidly identifying possible biomarkers, but so far, none have been approved by the U.S. Food and Drug Administration for clinical implementation. A major bottleneck seems to lie at the candidate verification step. To address this challenge, Steven Carr and co-workers at the Broad Institute, Massachusetts General Hospital, and Brigham and Women’s Hospital have developed multiple-reaction monitoring (MRM) assays that are coupled with stable-isotope dilution (SID) MS. To demonstrate the method, they used it to assess the levels of several cardiac markers.

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Heart MRMer. Researchers show that MRM assays can provide quantitative information for cardiac markers, such as C-reactive protein and cardiac troponin T.

Although immunoassays are the current gold standard for verification, antibody reagents do not exist for many of the candidate biomarkers identified in proteomics studies. A few research groups are developing MRM-SID assays as alternatives to antibody-based tests. MRM-SID assays have several advantages over immunoassays: they are more easily multiplexed, less costly, and can be developed much more quickly.

In this study, MRM-SID assays were developed for six proteins known to be clinically relevant to cardiac injury. Two to five of the highest responding peptides were analyzed for each protein, and three transitions were monitored for each peptide. The limit of detection and limit of quantitation for most proteins were in the low nanogram-per-milliliter range. The assays were reproducible: typical CVs were <10% for intra-assay variability and <25% for interassay variability.

Finally, the assays were applied to plasma samples taken at various time points from six patients undergoing planned myocardial infarctions (heart attacks). The plasma samples were depleted of high-abundance proteins with IgY antibodies, digested with trypsin, fractionated with strong-cation exchange chromatography, and analyzed by MRM-SID. Quantitative information was obtained for six of the nine target proteins. Levels of the monitored peptides followed similar trends from patient to patient throughout the experiment. The researchers say that these studies demonstrate that MRM assays are ready to step in and ease the bottleneck in the biomarker pipeline. (Mol. Cell. Proteomics 2009, DOI 10.1074/mcp.M900140-MCP200)