( Overview ...)Multiplexed Quantitative Peptide Assays for Protein Biomarkers of Cardiovascular Disease in Human Plasma

Overview

As the study of protein biomarkers increases in importance, extensive lists of candidate markers are being developed based on protein pathway information and discovery-based proteomics experiments. Even larger numbers of candidate genomic markers exist from DNA transcriptional profiling based on microarray-based gene expression studies or other genomic information.

While these genomic markers can be validated at the RNA level using real time PCR assays such as TaqMan kits, and at the protein level using Western blots, validation at the protein level requires specific antibodies to each protein which is a very time consuming and expensive task if hundreds of markers are of interest. A much higher throughput and more universal strategy is needed to narrow the huge number of candidate biomarkers that are being generated by both genomic and proteomics today at the protein level. The MIDAS workflow combined with multiplexed high throughput quantitative MRM assays on a MIDAS TRAQ system addresses these challenges.

Many technical challenges exist in the preliminary validation of putative protein biomarkers, such as the detection of low abundance proteins in complex tissue or biological fluids, as well as high-throughput, high precision quantitation. Validation of these markers for clinical use will require analysis of extensive candidate marker panels in thousands of clinical serum and plasma specimens. In this study, an assay was developed using multiple reaction monitoring (MRM) and MIDAS workflow on the MIDAS TRAQ system to detect and quantitate selected tryptic peptides, where each peptide represents a specific candidate protein biomarker identified for cardiovascular disease.

This application note demonstrates the specificity, sensitivity and reproducibility of this MRM based strategy in human plasma. A number of strategies were employed to develop high
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