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Single Isotopologue for In-Sample Calibration and Absolute Quantitation by LC-MS/MS.

Soroush Torkamannejad1, Ge Chang1, Fabusuyi A Aroge2

  • 1Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A1S6, Canada.

Journal of Proteome Research
|March 6, 2024
PubMed
Summary
This summary is machine-generated.

This study introduces a simplified mass spectrometry (MS) method using endogenous peptides for accurate quantification. The heavy matching light (HML) technique enhances biomarker discovery by simplifying calibration in complex biological samples.

Keywords:
absolute protein quantificationdigestion and recovery efficiencyin-sample calibrationmembrane proteinsparallel reaction monitoring (PRM)stable isotope dilutionsurrogate analyte methodsurrogate matrix methodtargeted mass spectrometry

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Area of Science:

  • Biomarker discovery
  • Analytical chemistry
  • Mass spectrometry (MS)

Background:

  • Targeted mass spectrometry (MS)-based absolute quantitative analysis is crucial for biomarker discovery.
  • Current methods rely on complex calibration using multiple isotopologues for surrogates and internal standards.
  • Accurate in-sample calibration is enabled by MS's precise mass measurement capabilities.

Purpose of the Study:

  • To simplify in-sample calibration in mass spectrometry (MS)-based absolute quantitative analysis.
  • To introduce a novel method, "heavy matching light" (HML), for direct quantification of endogenous analytes.
  • To assess the applicability of the HML method for proteins, peptides, and small molecules.

Main Methods:

  • Utilized endogenous light peptides as internal standards for simplified calibration.
  • Developed a mathematical deduction, "heavy matching light" (HML), for direct quantification.
  • Applied the HML method to quantify heart tissue digestion and recovery efficiency using spiked proteins.

Main Results:

  • The HML method provides essential assay performance parameters (LLOQ, calibration curve intercept) within the authentic matrix.
  • Quantification requires only a single isotopologue of the analyte, simplifying the process.
  • Demonstrated the assay's robustness in quantifying protein digestion and recovery efficiency.

Conclusions:

  • The HML method offers a simplified and robust approach for absolute quantitative analysis using targeted mass spectrometry (MS).
  • This technique is versatile and applicable to a wide range of analytes including proteins, peptides, and small molecules.
  • The method enhances biomarker discovery by streamlining calibration and improving assay performance in complex biological matrices.