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Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative
Susan E Abbatiello1, Birgit Schilling2, D R Mani1
1From the Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142;
Molecular & Cellular Proteomics : MCP
|February 20, 2015
Summary
This study shows that 11 labs can use liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) to accurately measure 125 peptides from 34 proteins in human plasma. This method ensures reliable protein quantification for biological and clinical research.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Clinical Medicine
Background:
- Robust protein quantification in biological samples is crucial for advancements in biology and clinical medicine.
- Previous work established liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) with isotope dilution for quantifying fewer proteins in plasma.
- Extending this, the study aimed to validate a highly multiplexed approach for a larger number of proteins.
Purpose of the Study:
- To demonstrate the feasibility of developing and applying highly multiplexed LC-MRM-MS assays across multiple laboratories.
- To quantify 125 peptides from 27 cancer-relevant and seven control proteins in human plasma.
- To establish analytical performance metrics and ensure reproducibility and precision in a multi-laboratory setting.
Main Methods:
- Development and validation of highly multiplexed LC-MRM-MS assays targeting 125 peptides.
- Implementation of a system suitability protocol (SSP) for real-time performance monitoring.
- Use of immunoaffinity depletion for abundant plasma proteins and stable isotope-labeled proteins as internal standards.
Main Results:
- Eleven laboratories successfully developed and applied the assays using 14 LC-MS systems.
- Achieved low to sub-nanogram/mL sensitivity for proteins in plasma.
- Demonstrated median inter-laboratory reproducibility below 20%, suitable for biomarker verification.
- Precisely and reproducibly quantified analytes in blinded samples simulating an inter-laboratory clinical study.
Conclusions:
- Highly multiplexed LC-MRM-MS with stable isotope dilution is a sensitive, specific, and reproducible method for protein quantification in complex matrices like plasma.
- The system suitability protocol is effective for ensuring data quality and monitoring assay performance.
- This approach is validated for inter-laboratory studies, supporting its use in clinical research and biomarker verification.
