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.

Fig. 1

Strategy for generation of peptide (A) and protein (B) standard addition curves in plasma for SISCAPA assay development of cTnI and IL-33.

Fig. 2. Peptide response curves before and after antibody enrichment. Peak area ratios of analyte (¹²C-peptide) and internal standard (¹³C-peptide) were determined from the XIC of the most abundant transition ions (cTnI [¹²C]623.3/1018.5, [¹³C]626.3/1024.5; IL-33 Pep-1 [¹²C]516.8/816.5, [¹³C]519.3/821.5; IL-33 Pep-2 [¹²C]794.4/1084.9, [¹³C]796.0/1087.5)

The observed concentration value was plotted against the expected concentration value for each individual response curve before (dashed line) and after (solid line) antibody enrichment. The average LOD is labeled at the bottom of the linear range of each antibody–peptide curve. Enrichment factors between 2000 (IL-33 Pep-2) and 8000 (cTnI) were calculated as the ratio of LOD observed before and after antibody enrichment.

Fig. 3. Reproducibility of multiplexed SISCAPA assay for cTnI and IL-33

Four independent protein standard addition curves for the proteins are shown. Target peptides were enriched once in separate tubes (uniplex) or 3 times in 1 tube (multiplex). The average protein concentration of 3 injections (uniplex) or 12 injections (multiplex) for cTnI (A) and IL-33 Pep-1 (B) was plotted against the theoretical protein concentration.

Fig. 4. Correlation of cTnI concentrations determined by SISCAPA and immunoassay in PMI patient plasma

The linear fit obtained using Deming regression of the correlation between commercially available immunoassay kit and SISCAPA of cTnI concentration on PMI patient time course samples measured >1 μg/L by both assays. The equation of the robust linear regression line is displayed with the Pearson coefficient of correlation (R) calculated for the comparison of these 2 methods. At concentrations <1 μg/L for cTnI, the interference of background signal produced a nonlinear trend; these data points were therefore excluded from the comparison.

Fig. 5. cTnI concentrations determined by SISCAPA in PMI patient plasma

Plasma concentration of cTnI was measured in 5 patients undergoing PMI. Six time points were sampled over the course of the procedure (0, 0.2, 1, 2, 4, and 24 h). Each measurement is an average of 3 injections; error bars represent SD. A nonlinear scale was used for the time axis.