Quantification of cardiac troponin I in human plasma by immunoaffinity enrichment and targeted mass spectrometry
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Quantification of cardiac troponin I (cTnI), a protein biomarker used for diagnosing myocardial infarction, has been achieved in native patient plasma based on an immunoaffinity enrichment strategy and isotope dilution (ID) liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The key steps in the workflow involved isolating cTnI from plasma using anti-cTnI antibody coupled to magnetic nanoparticles, followed by an enzymatic digestion with trypsin. Three tryptic peptides from cTnI were monitored and used for quantification by ID-LC-MS/MS via multiple reaction monitoring (MRM). Measurements were performed using a matrix-matched calibration system. NIST SRM 2921 Human Cardiac Troponin Complex acted as the calibrant and a full-length isotopically labeled protein analog of cTnI was used as an internal standard. The method was successfully demonstrated on five patient plasma samples, with cTnI concentrations measuring between 4.86 μg/L and 11.3 μg/L (signifying moderate myocardial infarctions). LC-MS/MS measurement precision was validated by three unique peptides from cTnI and two MRM transitions per peptide. Relative standard deviation (CV) from the five plasma samples was determined to be ≤14.3%. This study has demonstrated that quantification of cTnI in native plasma from myocardial infarction patients can be achieved based on an ID-LC-MS/MS method. The development of an ID-LC-MS/MS method for cTnI in plasma is a first step for future certification of matrix-based reference materials, which may be used to help harmonize discordant cTnI clinical assays.
KeywordsProtein biomarker Cardiac troponin I Mass spectrometry Isotope dilution Quantification Multiple reaction monitoring
The authors thank Dr. Andrew Hoofnagle from the University of Washington for providing clinical samples and assistance, and Dr. Eric Kilpatrick from the National Institute of Standards and Technology for his logistical help receiving the patient plasma samples and for his scientific discussions. We also acknowledge the support of the Professional Research Experience Program (PREP) through the University of Maryland, College Park and the National Institute of Standards and Technology.
Compliance with ethical standards
Patient plasma samples were obtained as de-identified, residual clinical samples from the University of Washington. All work with human derived samples was reviewed and approved by the NIST Human Subjects Protection Office.
Conflict of interest
The authors declare that they have no conflicts of interest.
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