Highly multiplexed quantification by targeted mass spectrometry to triage protein candidate biomarkers in plasma

Despite improvements in capabilities of proteomics technologies, the introduction of new plasma-based protein biomarkers for clinical use remains low. One reason is the cumbersome requirement to test thousands of protein candidates in follow-up quantitative verification studies. We sought to evaluate internal standard triggered parallel reaction monitoring (IS-PRM) in the context of biomarker verification by developing a method to quantify 5,176 peptides (1,314 proteins) as candidate biomarkers for early detection of breast cancer. Method performance was characterized in a response curve showing large linear range (4 orders of magnitude) and good repeatability (median CV 7.7%). The method was applied to pools of cancer and control human plasma, detecting 893 proteins and qualifying 164 candidates to advance for further evaluation. The method shows good quantitative performance, greatly expanding the capabilities for quantification of large numbers of proteins, and is well suited for large scale relative quantification of protein sets.