Quantification of cardiovascular biomarkers in patient plasma by targeted mass spectrometry and stable isotope dilution

Abstract

Verification of candidate biomarkers requires specific assays to selectively detect and quantify target proteins in accessible biofluids. The primary objective of verification is to screen potential biomarkers to ensure that only the highest quality candidates from the discovery phase are taken forward into preclinical validation. Because antibody reagents for a clinical grade immunoassay often exist for a small number of candidates, alternative methodologies are required to credential new and unproven candidates in a statistically viable number of serum or plasma samples. Using multiple reaction monitoring coupled with stable isotope dilution MS, we developed quantitative, multiplexed assays in plasma for six proteins of clinical relevance to cardiac injury. The process described does not require antibodies for immunoaffinity enrichment of either proteins or peptides. Limits of detection and quantitation for each signature peptide used as surrogates for the target proteins were determined by the method of standard addition using synthetic peptides and plasma from a healthy donor. Limits of quantitation ranged from 2 to 15 ng/ml for most of the target proteins. Quantitative measurements were obtained for one to two signature peptides derived from each target protein, including low abundance protein markers of cardiac injury in the nanogram/milliliter range such as the cardiac troponins. Intra- and interassay coefficients of variation were predominantly <10 and 25%, respectively. The configured multiplex assay was then used to measure levels of these proteins across three time points in six patients undergoing alcohol septal ablation for hypertrophic obstructive cardiomyopathy. These results are the first demonstration of a multiplexed, MS-based assay for detection and quantification of changes in concentration of proteins associated with cardiac injury in the low nanogram/milliliter range. Our results also demonstrate that these assays retain the necessary precision, reproducibility, and sensitivity to be applied to novel and uncharacterized candidate biomarkers for verification of proteins in blood.

Fig. 1.

Assay configuration and sample preparation work flow. Work flow A represents the method used to select signature peptides for proteins associated with cardiac injury. Work flow B represents assay configuration conducted in parallel for MS instrument optimization and peptide separation by SCX. Work flow C represents the plasma processing and limited fractionation/MRM assay used for all six patients and time points (baseline and 4 and 24 h post injury). Three technical replicates and two process replicates for all samples were performed. IS, internal standard.

Fig. 2.

Quantitation of moderately abundant proteins in patient plasma samples. Line plots of log(concentration) versus time of CRP (A) and MRP14 (B) at baseline and 4 and 24 h post injury as measured by MRM assay with two different signature peptides for each protein across six patients. Pink and blue traces indicate two different process replicates for each patient and time point.

Fig. 3.

Quantitation of low abundance proteins in patient plasma samples. Line plots of log(concentration) versus time of MPO (A), cTnI (B), cTnT (C), and NT-proBNP (D) at baseline and 4 and 24 h post injury as measured by MRM assay with one representative signature peptide for each protein across six patients. Pink and blue traces indicate two different process replicates for each patient and time point.

Fig. 4.

Comparison of MRM assay with clinical immunoassay. Rank regression analysis of protein concentrations obtained by immunoassay of non-depleted plasma samples and values obtained by MRM for CRP (A), MPO (B), cTnT (C), and NT-proBNP (D) is shown. Spearman rank correlation (R) is calculated for each peptide/protein.