HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: clinchem.2009.123935v1 55/6/1108    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Google Scholar Articles by Kuhn, E. Articles by Carr, S. A. PubMed PubMed Citation Articles by Kuhn, E. Articles by Carr, S. A.

Developing Multiplexed Assays for Troponin I and Interleukin-33 in Plasma by Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry

¹ Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA;² Cardiovascular Division, Brigham and Women’s Hospital, Boston, MA;³ Cardiology Division and Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Boston, MA.

^aAddress correspondence to this author at: Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142. Fax 617-252-1902; e-mail scarr{at}broad.mit.edu.

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 µg/L, with imprecision <13% for both proteins, processed individually or multiplexed. Results correlated well (R = 0.89) with a commercial immunoassay.

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 µg/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. .

The following articles in journals at HighWire Press have cited this article:

				H. Keshishian, T. Addona, M. Burgess, D. R. Mani, X. Shi, E. Kuhn, M. S. Sabatine, R. E. Gerszten, and S. A. Carr Quantification of Cardiovascular Biomarkers in Patient Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution Mol. Cell. Proteomics, October 1, 2009; 8(10): 2339 - 2349. [Abstract] [Full Text] [PDF]