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Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification
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Absolute Quantitative Targeted Proteomics Assays for Plasma Proteins.

Yassene Mohammed1,2, David Goodlett3,4,5, Christoph H Borchers6,7,8,9

  • 1Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands. y.mohammed@lumc.nl.

Methods in Molecular Biology (Clifton, N.J.)
|February 13, 2023
PubMed
Summary

This study presents a rapid, 1-hour targeted proteomics protocol for absolute blood plasma protein quantification. The method uses mass spectrometry and heavy-labeled standards, offering a cost-effective, sensitive alternative for biomarker monitoring.

Keywords:
Blood plasma proteinsLongitudinal analysisQuantitative evaluationTargeted proteomics

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Proteomics

Background:

  • Preclinical and clinical trials demand high-throughput, precise analytical methods for complex sample characterization and biomarker monitoring.
  • Targeted proteomics offers a solution for quantifying protein abundances in biological matrices, particularly blood plasma.
  • Existing methods like ELISA can be time-consuming and less sensitive compared to mass spectrometry-based approaches.

Purpose of the Study:

  • To describe a protocol for absolute quantification of proteins in blood plasma using targeted multiple reaction monitoring (MRM) proteomics.
  • To enable rapid, robust, and high-throughput biomarker monitoring with minimal sample consumption.
  • To provide a cost-effective and sensitive alternative to traditional methods like ELISA.

Main Methods:

  • Utilized mass spectrometry (MS)-based targeted proteomics employing multiple reaction monitoring (MRM) with stable isotope-labeled internal standard (SIS) peptides.
  • Developed a 270-protein panel for comprehensive plasma proteome characterization.
  • Employed heavy-labeled internal standards for absolute protein quantification.

Main Results:

  • Achieved absolute quantitative proteomic characterization of blood plasma within a 1-hour gradient.
  • Demonstrated the protocol's effectiveness with non-depleted plasma, simplifying implementation.
  • Validated the method's compatibility with common blood plasma collection methods (K2EDTA and sodium citrate).

Conclusions:

  • The described targeted MRM proteomics protocol provides a rapid, precise, and robust method for absolute blood plasma protein quantification.
  • This approach facilitates high-throughput biomarker monitoring in preclinical and clinical settings.
  • The protocol is user-friendly, cost-effective, and adaptable to standard laboratory practices.