Comparative evaluation of peptide vs. protein-based calibration for quantification of cardiac troponin I using ID-LC-MS/MS

Objectives: An analytical protocol based on isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS), which includes a peptide-based calibration strategy, was developed and validated for the determination of cardiac troponin I (cTnI) levels in clinical samples. Additionally, the developed method was compared with a protein-based calibration strategy, using cTnI serving as a model for low-abundant proteins. The aim is to evaluate new approaches for protein quantification in complex matrices, supporting the metrology community in implementing new methods and developing fit-for-purpose SI- traceable peptide or protein primary calibrators. Methods: To establish traceability to SI units, peptide impurity correction amino acid analysis (PICAA) was conducted to determine the absolute content of signature peptides in the primary standards. Immunoaffinity enrichment was used to capture cTnI from human serum, with a comparison between microbeads and nanobeads to improve enrichment efficiency. Parallel reaction monitoring was used to monitor two signature peptides specific to cTnI. Various digestion parameters were optimized to achieve complete digestion. Results: The analytical method demonstrated selectivity and specificity, allowing the quantification of cTnI within 0.9–22.0 μg/L. The intermediate precision RSD was below 28.9 %, and the repeatability RSD was below 5.8 % at all concentration levels, with recovery rates ranging from 87 % to 121 %. The comparison of calibration strategies showed similar LOQ values, but the peptide-based calibration exhibited significant quantitative bias in recovery rates. The data are available via ProteomeXchange (PXD055104). Conclusions: This isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method, based on peptide calibration, successfully quantified cTnI in human serum. Comparing this with protein-based calibration highlighted both the strengths and potential limitations of peptide-based strategies.