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Internal Standard Triggered-Parallel Reaction Monitoring Mass Spectrometry Enables Multiplexed Quantification of
Jacob J Kennedy1, Jeffrey R Whiteaker1, Richard G Ivey1
1Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, United States.
Analytical Chemistry
|June 29, 2022
Summary
This study introduces an improved assay for plasma biomarker discovery, enabling the testing of thousands of candidates. The new method enhances quantification and prioritizes breast cancer biomarkers for clinical validation.
Area of Science:
- Biochemistry
- Proteomics
- Clinical Diagnostics
Background:
- Clinical translation of plasma biomarkers is hindered by the costly validation process and limited multiplexing capabilities of current assays.
- Existing mass spectrometry methods for biomarker discovery offer poor quantitative performance and are restricted to a few hundred proteins.
Purpose of the Study:
- To develop and validate a novel assay for high-throughput quantification of plasma biomarker candidates.
- To improve the prioritization of potential biomarkers for clinical validation in breast cancer.
Main Methods:
- Coupling immunodepletion and fractionation with an internal standard triggered-parallel reaction monitoring (IS-PRM) assay.
- Quantification of 5176 peptides representing 1314 breast cancer biomarker candidates in human plasma.
- Characterization of assay precision, linearity, and sensitivity.
Main Results:
- The IS-PRM assay demonstrated high precision (median % CV of 7.7%), linearity (median R² > 0.999), and sensitivity (median LLOQ < 1 fmol).
- Quantified 893 proteins across patient and control plasma pools.
- Verified 162 candidate biomarkers in cancer pools, with 22 verified in all three cancer pools.
Conclusions:
- The developed IS-PRM assay significantly expands the capacity for quantifying large numbers of proteins in plasma.
- This assay is well-suited for efficient prioritization of viable candidate biomarkers for clinical validation, addressing a key bottleneck in biomarker discovery.
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