Christoph Borchers, Ph.D., facility director of the University of Victoria Genome BC Proteomics Centre, presented his concept for developing biomarkers that predict risk for inflammatory bowel disease (IBD) and cardiovascular disease (CVD) using multireaction monitoring (MRM) for quantitative measurement of proteins.
The range of clinically significant proteins in human blood and serum spans as much as 12 orders of magnitude and is a serious challenge to most protein biomarker screening technologies. MRM can provide accurate quantitation of proteins over a large dynamic range within a single sample. Absolute quantitation of proteins is made possible by the use of isotopically coded peptides as standards.
According to Dr. Borchers, each protein can have many peptides being used for MRM analysis associated with it. By developing an assay that includes the standard peptide spiked in at a ratio of approximately 1:1 to the endogenous peptide, it is possible to cover a large portion of the range of abundance of proteins in the human blood or serum proteome. Dr. Borchers and his colleagues then created cocktails of these standards tailored to specific diseases for large-scale proteomic analysis.
In the case of cardiovascular disease, Dr. Borchers identified approximately 80 proteins that are known to be potential cardiovascular risk biomarkers. In a preliminary study using a standard cocktail, he narrowed the list down to five markers that could stratify patient groups with and without CVD with 80–90% accuracy.
Taking these techniques into IBD, Dr. Borchers is looking at approximately 100 proteins that are potential markers of risk for the disease. Diagnosis of IBD is complicated by the flare and remission cycle, which obscures endpoints and at times generates large placebo effects in clinical trials. Reliable biomarkers and companion diagnostics would eliminate much of the uncertainty in IBD therapeutic discovery and development.
Progress in biomarker discovery in areas such as CVD and IBD depends on rapid, accurate methods for screening samples. “We’re talking about more than three million people being screened every year,” said Dr. Borchers.
“We need to have high throughput in addition to absolute quantitation. For this purpose we are now setting up an assay based on the immuno MALDI (iMALDI) technology we have pioneered. iMALDI combines immuno-affinity enrichment of peptides, MALDI mass spectrometry, and MRM. It is capable of analyzing a sample in a few seconds, or thousands of samples in one day, with absolute quantitative results for proteins.”