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

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.