Developing multiplexed assays for troponin I and interleukin-33 in plasma by peptide immunoaffinity enrichment and targeted mass spectrometry.
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 <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 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.
Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142, USA.
Eric
E
Kuhn
Terri
T
Addona
Hasmik
H
Keshishian
Michael
M
Burgess
D R
DR
Mani
Richard T
RT
Lee
Marc S
MS
Sabatine
Robert E
RE
Gerszten
Steven A
SA
Carr
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D000067596:Interleukin-33; D007378:Interleukins / Q000097:blood*; D010455:Peptides / Q000737:chemistry; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D019210:Troponin I / Q000097:blood*
C506536:IL33 protein, human; D000067596:Interleukin-33; D007378:Interleukins; D010455:Peptides; D019210:Troponin I