The development of clinical biomarkers involves discovery, verification, and validation. Recently, multiple reactionmonitoring (MRM) coupled with stable isotope dilution mass spectrometry (IDMS) has shown considerable promise for the directquantification of proteins in clinical samples. In particular, multiple biomarkers have been tracked in a single experiment usingMRM-based MS approaches combined with liquid chromatography. We report here a highly reproducible, quantitative, anddynamic MRM system for validating multi-biomarker proteins using Nanoflow HPLC-Microfluidics Chip/Triple-QuadrupoleMS. In this system, transitions were acquired only during the retention window of each eluting peptide. Transitions with the highestMRM-MS intensities for the five target peptides from colon cancer biomarker candidates were automatically selected usingOptimizer software. Relative to the corresponding non-dynamic system, the dynamic MRM provided significantly improvedcoefficients of variation in experiments with large numbers of transitions. Linear responses were obtained with concentrationsranging from fmol to pmol for five target peptides.
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