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

ABSTRACT: 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.

SUBMITTER: Kuhn E

PROVIDER: S-EPMC2865473 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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

Kuhn Eric E Addona Terri T Keshishian Hasmik H Burgess Michael M Mani D R DR Lee Richard T RT Sabatine Marc S MS Gerszten Robert E RE Carr Steven A SA

Clinical chemistry 20090416 6

<h4>Background</h4>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 ...[more]

PMID: 19372185

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Project description:Oral cancer is one of the most common cancers worldwide, and there are currently no biomarkers approved for aiding its management. Although many potential oral cancer biomarkers have been discovered, very few have been verified in body fluid specimens in parallel to evaluate their clinical utility. The lack of appropriate multiplexed assays for chosen targets represents one of the bottlenecks to achieving this goal. In the present study, we develop a peptide immunoaffinity enrichment-coupled multiple reaction monitoring-mass spectrometry (SISCAPA-MRM) assay for verifying multiple reported oral cancer biomarkers in saliva. We successfully produced 363 clones of mouse anti-peptide monoclonal antibodies (mAbs) against 36 of 49 selected targets, and characterized useful mAbs against 24 targets in terms of their binding affinity for peptide antigens and immuno-capture ability. Comparative analyses revealed that an equilibrium dissociation constant (KD ) cut-off value < 2.82 × 10-9 m could identify most clones with an immuno-capture recovery rate >5%. Using these mAbs, we assembled a 24-plex SISCAPA-MRM assay and optimized assay conditions in a 25-μg saliva matrix background. This multiplexed assay showed reasonable precision (median coefficient of variation, 7.16 to 32.09%), with lower limits of quantitation (LLOQ) of <10, 10-50, and >50 ng/ml for 14, 7 and 3 targets, respectively. When applied to a model saliva sample pooled from oral cancer patients, this assay could detect 19 targets at higher salivary levels than their LLOQs. Finally, we demonstrated the utility of this assay for quantification of multiple targets in individual saliva samples (20 healthy donors and 21 oral cancer patients), showing that levels of six targets were significantly altered in cancer compared with the control group. We propose that this assay could be used in future studies to compare the clinical utility of multiple oral cancer biomarker candidates in a large cohort of saliva samples.

Dataset Information

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

Publications

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

Similar Datasets

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