Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution

Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothe-sis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target pro-teins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable iso-tope dilution mass spectrometry, we describe here the de-velopment of quantitative, multiplexed assays for six pro-teins in plasma that achieve limits of quantitation in the 1–10 ng/ml range with percent coefficients of variation from 3 to 15 % without immunoaffinity enrichment of either protein