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

Abstract

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.

Development of an Absolute Quantification Method for hERG Using PRM with Single Isotopologue in-Sample Calibration

“…Matrix effect is paramount to the assay performance, including the sensitivity, LLOQ, and accuracy . The most appropriate evaluation of different candidate transitions requires the use of an authentic matrix.…”

“…For the PRM analysis, two in-sample calibration strategies were used. First was the recently developed HML approach, in which synthetic heavy peptides were spiked in a sample at known concentrations as calibrators, and the corresponding endogenous hERG peptides were used as internal standards to construct calibration curves. Second was the classic SA approach, in which synthetic light peptides from hERG were spiked in the sample at known concentrations as the calibrators, and the corresponding heavy synthetic peptides were also spiked in the sample as the internal standards to construct the calibration curves.…”

“…The Q Exactive HF parameters for PRM analysis were MS1 scan with a m / z range of 400–2000, a mass resolution of 120,000, an AGC target of 3 × 10 6 , and a maximum ion injection time of 200 ms. For the MS2 scans, the parameters were as follows: mass resolution, 30,000; AGC target, 2 × 10 5 ; maximum ion injection time, 150 ms; and isolation window, 2.0 Th, with a 0.5 Th offset to include additional isotopic peaks, as previously reported . The MS2 scans were conducted based on the inclusion list that included the m / z ratio of the endogenous light and the corresponding heavy reference peptides of hERG and a duration of more than 10 min scheduled around the elution peaks of every target peptide.…”

“…To calculate the endogenous peptide concentrations, the methods used for SA and HML were different. For HML analysis, the concentration of endogenous peptides was calculated by 1 − i n t e r c e p t normals normall normalo normalp normale as described in a previous publication . For SA analysis, the concentration was obtained by normali normaln normalt normale normalr normalc normale normalp normalt normals normall normalo normalp normale .…”

“…To provide a useful tool for studying hERG, we develop here a MS-based and targeted parallel reaction monitoring (PRM) assay for the absolute quantification of hERG. Our assay takes advantage of the recently published single-isotopologue in-sample calibration approach that uses heavy-match-light (HML) mathematical treatment to quantify the endogenous analyte. We also compared the analysis results with those of the classic standard addition (SA) in-sample calibration approach.…”