Triage of >1300 candidate biomarkers in plasma using internal standard-triggered parallel reaction monitoring mass spectrometry

Despite advances in proteomic technologies, clinical translation of plasma biomarkers remains low, partly due to a major bottleneck between the discovery of hundreds of candidate biomarkers and downstream costly clinical validation studies. Due to a dearth of multiplexable assays, generally only a few candidate biomarkers are tested, and the validation success rate is accordingly low. Here, we demonstrate the capability of internal standard triggered parallel reaction monitoring (IS-PRM) to prioritize candidate biomarkers for validation studies. A 5,176-plex IS-PRM assay was developed and implemented in human plasma to quantify peptides representing 1,314 breast cancer biomarker candidates. Compared to prior approaches using data-dependent analysis, IS-PRM showed improved sensitivity and precision and enabled rank-ordering of candidate biomarkers for validation studies. The method greatly expands the capabilities for quantification of large numbers of proteins and is well suited for large-scale prioritization of viable candidate biomarkers.