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Single Isotopologue for In-Sample Calibration and Absolute Quantitation

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Targeted mass spectrometry (MS)-based absolute quantitative analysis has been increasingly used in biomarker discovery. The ability to accurately measure the masses by MS enabled the use of isotope-incorporated surrogates having virtually identical physiochemical properties of the target analytes as calibrators. Such a unique capacity allowed an accurate in-sample calibration. Current in-sample calibration uses multiple isotopologues or structural analogues for both the surrogate and the internal standard. Here, we simplified this common practice by using endogenous light peptides as the internal standards and used a mathematical deduction of “heavy matching light, HML” to directly quantify endogenous analyte. This method provides all necessary assay performance parameters in the authentic matrix, including the lower limit of quantitation (LLOQ) and intercept of the calibration curve, by using only a single isotopologue of the analyte. This method can be applied to the quantitation of proteins, peptides, and small molecules. Using this method, we quantified the efficiency of heart tissue digestion and recovery using sodium deoxycholate as a detergent and two spiked exogenous proteins as mimics of heart proteins. The results demonstrated the robustness of the assay.
The fulltext of this paper is embargoed until March 6, 2025 due to the embargo policies of the American Chemical Society. If you need fulltext access to this paper immediately, please contact with your request.
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Accepted date
6 March 2024
Available date
6 March 2025
DOI: 10.1021/acs.jproteome.3c00848
Publication details
Publication title
Journal of Proteome Research
Document title
Single Isotopologue for In-Sample Calibration and Absolute Quantitation by LC-MS/MS
American Chemical Society
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Copyright is held by the publisher with many rights continuing to also be held by the author(s).
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