|Better biomarkers are urgently needed to improve diagnosis, guide molecularly targeted therapy, and monitor activity and therapeutic response across a wide spectrum of disease. Proteomics methods based on mass spectrometry hold special promise for the discovery of novel biomarkers that might form the foundation for new clinical blood tests, but to date their contribution to the diagnostic armamentarium has been disappointing. Advances in methods and technology now enable conceptualization and evaluation of a comprehensive biomarker pipeline from the following essential process components: candidate discovery in tissues or proximal fluids, prioritization of candidates using targeted screening approaches in plasma, verification of candidates by quantitative MS-based assays in patient (>100) plasma, biomarker validation in very large population studies, and commercialization. |
This talk will focus on the discovery-through-quantitative verification steps with examples from our ongoing studies in breast cancer and cardiovascular disease.
Lack of robust quantitative methods with sufficient sensitivity, reproducibility and throughput has significantly hampered our ability to credential candidates coming from unbiased proteomic discovery efforts since useful Ab reagents for the vast majority do not exist. My laboratory has focused considerable effort in addressing this serious barrier by developing robust targeted assay methods employing mass spectrometry to screen and quantify low abundance proteins in plasma. We have recently demonstrated that multiplexed assays for proteins at the low ng/mL level in plasma can be configured. Further improvements will come from the use of peptide immunoaffinity enrichment, referred to as SISCAPA, which holds particular promise for simplifying sample preparation and increasing both throughput and sensitivity of MRM-based assays. Using SISCAPA, assays can be readily configured that enable quantitation of proteins present at low ng/mL levels directly from plasma.