Quantification of cardiac troponin I in human plasma by immunoaffinity enrichment and targeted mass spectrometry

Nicole A. Schneck, Karen W. Phinney, Sang Bok Lee, Mark S. Lowenthal

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. Graphical abstract A schematic of the workflow for measuring cardiac troponin I (cTnI), a low-abundant protein biomarker used for diagnosing myocardial infarction, in human plasma by isotope-dilution LC-MS/MS analysis.