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

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Abstract

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.

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.

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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, 20742, USA

    Nicole A. Schneck & Sang Bok Lee

  2. Biomolecular Measurement Division, National Institute of Standards and Technology, 100 Bureau Drive, Stop 8314, Gaithersburg, MD, 20899, USA

    Nicole A. Schneck, Karen W. Phinney & Mark S. Lowenthal

Authors
  1. Nicole A. Schneck
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  2. Karen W. Phinney
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  3. Sang Bok Lee
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  4. Mark S. Lowenthal
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Corresponding authors

Correspondence to Sang Bok Lee or Mark S. Lowenthal.

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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.

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The authors declare that they have no conflicts of interest.

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Certain commercial equipment, instruments, and materials are identified in this paper to specify the experimental procedures and analytical methods adequately. In no case does such identification imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment are necessarily the best available for the purpose.

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Schneck, N.A., Phinney, K.W., Lee, S.B. et al. Quantification of cardiac troponin I in human plasma by immunoaffinity enrichment and targeted mass spectrometry. Anal Bioanal Chem 410, 2805–2813 (2018). https://doi.org/10.1007/s00216-018-0960-7

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  • DOI: https://doi.org/10.1007/s00216-018-0960-7

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