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Clin Chem DOI:10.1373/clinchem.2009.123935

Developing multiplexed assays for troponin I and interleukin-33 in plasma by peptide immunoaffinity enrichment and targeted mass spectrometry.

Publication TypeJournal Article
Year of Publication2009
AuthorsKuhn, E, Addona, T, Keshishian, H, Burgess, M, Mani, DR, Lee, RT, Sabatine, MS, Gerszten, RE, Carr, SA
JournalClin Chem
Date Published2009 Jun
KeywordsAmino Acid Sequence, Chromatography, Affinity, Humans, Interleukin-33, Interleukins, Peptides, Sensitivity and Specificity, Tandem Mass Spectrometry, Troponin I

BACKGROUND: Protein biomarker candidates from discovery proteomics must be quantitatively verified in patient samples before they can progress to clinical validation. Here we demonstrate that peptide immunoaffinity enrichment coupled with stable isotope dilution mass spectrometry (SISCAPA-MRM) can be used to configure assays with performance suitable for candidate biomarker verification. As proof of principle, we configured SISCAPA assays for troponin I (cTnI), an established biomarker of cardiac injury, and interleukin 33 (IL-33), an emerging immunological and cardiovascular marker for which robust immunoassays are currently not available.

METHODS: We configured individual and multiplexed assays in which peptides were enriched from digested human plasma using antipeptide antibodies. Assay performance was established using response curves for peptides and proteins spiked into normal plasma. We quantified proteins using labeled peptides as internal standards, and we measured levels of cTnI in patients who underwent a planned myocardial infarction for hypertrophic obstructive cardiomyopathy.

RESULTS: Measurement of cTnI and IL-33 proteins from trypsin-digested plasma was linear from 1.5 to 5000 microg/L, with imprecision

CONCLUSIONS: We used an established biomarker of cardiac injury and an emerging biomarker to demonstrate how SISCAPA can detect and quantify changes in concentration of proteins present at 1-10 microg/L in plasma. Our results demonstrate that these assays can be multiplexed and retain the necessary precision, reproducibility, and sensitivity to be applied to new and uncharacterized candidate biomarkers for verification of low-abundance proteins in blood.


Alternate JournalClin. Chem.
PubMed ID19372185
PubMed Central IDPMC2865473
Grant ListR01 HL096738 / HL / NHLBI NIH HHS / United States
R01 HL096738-01 / HL / NHLBI NIH HHS / United States
U01-HL081341 / HL / NHLBI NIH HHS / United States
U01 HL081341 / HL / NHLBI NIH HHS / United States
U24 CA126476 / CA / NCI NIH HHS / United States
1U24 CA126476-02 / CA / NCI NIH HHS / United States