Internal Standard Triggered-Parallel Reaction Monitoring Mass Spectrometry Enables Multiplexed Quantification of Candidate Biomarkers in Plasma

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 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. Previously, mass spectrometry-based approaches
have been used to fill this gap but feature poor quantitative performance
and were generally limited to hundreds of proteins. Here, we demonstrate
the capability of an internal standard triggered-parallel reaction
monitoring (IS-PRM) assay to greatly expand the numbers of candidates
that can be tested with improved quantitative performance. The assay
couples immunodepletion and fractionation with IS-PRM and was developed
and implemented in human plasma to quantify 5176 peptides representing
1314 breast cancer biomarker candidates. Characterization of the IS-PRM
assay demonstrated the precision (median % CV of 7.7%), linearity
(median R2 > 0.999 over 4 orders of
magnitude),
and sensitivity (median LLOQ < 1 fmol, approximately) to enable
rank-ordering of candidate biomarkers for validation studies. Using
three plasma pools from breast cancer patients and three control pools,
893 proteins were quantified, of which 162 candidate biomarkers were
verified in at least one of the cancer pools and 22 were verified
in all three cancer pools. The assay greatly expands capabilities
for quantification of large numbers of proteins and is well suited
for prioritization of viable candidate biomarkers.