Development of an ID-LC–MS/MS method using targeted proteomics for quantifying cardiac troponin I in human serum

Meltem Asicioglu, Merve Oztug*, Nevin Gul Karaguler

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Background: Cardiac troponin is a complex protein consisting of the three subunits I, T and C located in heart muscle cells. When the heart muscle is damaged, it is released into the blood and can be detected. Cardiac troponin I (cTnI) is considered the most reliable and widely accepted test for detecting and confirming acute myocardial infarction. However, there is no current standardization between the commercial assays for cTnI quantification. Our work aims to create a measurement procedure that is traceable to the International System of Units for accurately measuring cardiac cTnI levels in serum samples from patients. Methods: The workflow begins with immobilizing anti-cTnI antibodies onto magnetic nanoparticles to form complexes. These complexes are used to isolate cTnI from serum. Next, trypsin is used to enzymatically digest the isolated cTnI. Finally, the measurement of multiple cTnI peptides is done simultaneously using isotope dilution liquid chromatography–tandem mass spectrometry (ID-LC–MS/MS). Results: The maximum antibody immobilization was achieved by combining 1 mg of nanoparticles with 100 μg of antibody, resulting in an average of 59.2 ± 5.7 μg/mg of immobilized antibody. Subsequently, the anti-cTnI-magnetic nanoparticle complex was utilized to develop and validate a method for quantifying cTnI in human serum using ID-LC–MS/MS and a protein calibration approach. The analytical method was assessed regarding linearity and recovery. The developed method enables the quantification of cTnI from 0.7 to 24 μg/L (R > 0.996). The limit of quantification was 1.8 μg/L and the limit of detection was 0.6 μg/L. Intermediate precision was ≤ 9.6% and repeatability was 2.0–8.7% for all quality control materials. The accuracy of the analyzed quality control materials was between 90 and 110%. Total measurement uncertainties for target value assignment (n = 6) were found to be ≤ 12.5% for all levels. Conclusions: The analytical method demonstrated high analytical performance in accurately quantifying cardiac troponin I levels in human serum. The proposed analytical method has the potential to facilitate the harmonization of cTnI results between clinical laboratories, assign target values to secondary certified reference materials and support reliable measurement of cTnI. Graphical Abstract: [Figure not available: see fulltext.].

Orijinal dilİngilizce
Makale numarası40
DergiClinical Proteomics
Basın numarası1
Yayın durumuYayınlandı - Ara 2023

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Publisher Copyright:
© 2023, BioMed Central Ltd., part of Springer Nature.


The authors would like to express their gratitude to Dr. Evren Saban from TÜBİTAK UME, Turkey, his guidance greatly contributed to the successful implementation of this research. We also thank Dr. Emrah Yurek from the Kanuni Sultan Suleyman Training and Research Hospital (Istanbul) for providing clinical samples. This project 18HLT10 CardioMet has received funding from the EMPIR programme co-financed by the Participating States and from the European Union’s Horizon 2020 research and innovation programme.

FinansörlerFinansör numarası
Kanuni Sultan Suleyman Training and Research Hospital
Horizon 2020 Framework Programme
European Metrology Programme for Innovation and Research
Türkiye Bilimsel ve Teknolojik Araştırma Kurumu

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