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Multiplexed triage of candidate biomarkers in plasma using internal standard triggered-parallel reaction monitoring mass spectrometry

By Jacob J Kennedy, Jeffrey R Whiteaker, Richard G Ivey, Aura Burian, Shrabanti Chowdhury, Chia-Feng Tsai, Tao Liu, ChenWei Lin, Oscar Murillo, Rachel A. Lundeen, Lisa A Jones, Philip R. Gafken, Gary Longton, Karin D Rodland, Steven Skates, John Landua, Pei Wang, Michael T Lewis, Amanda G. Paulovich

Posted 03 Sep 2021
bioRxiv DOI: 10.1101/2021.09.02.458791

Despite advances in proteomic technologies, clinical translation of plasma biomarkers remains low, partly due to a major bottleneck between the discovery 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 assay coupling immunodepletion and fractionation with IS-PRM 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 (912 vs 295 proteins quantified) and precision (CV 0.1 vs 0.27) enabling rank-ordering of candidate biomarkers for validation studies. The assay greatly expands capabilities for quantification of large numbers of proteins and is well suited for prioritization of viable candidate biomarkers.

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