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

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 with the target analytes as calibrators. Such a unique capacity allowed for 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 an 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.