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.

Methods for Peptide and Protein Quantitation by Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry

“…Our analyses of the CPTAC protein spike data from two sites yielded CVs ranging from 3-21% with a median of 8.5%. These results also are consistent with previous evaluations of SID (2,3,7,8).…”

“…In practice, this approach has proven sensitive enough to apply to challenging protein quantitation problems. For example, proteins can be quantified at singledigit copy numbers in cells (6) and in plasma at levels approaching ng/ml (7,8). With antibody-based enrichment, LC-MRM-MS can achieve even greater sensitivity (9 -12).…”

Applications of targeted proteomics in systems biology and translational medicine

“…Kuzyk and coworkers developed a mixture of 45 peptide standards in EDTA‐plasma without affinity depletion or enrichment and found that 31 of the 45 are putative markers of cardiovascular disease 116. Keshishian and coworkers developed quantitative assays without immunoaffinity enrichment for six proteins of clinical relevance to cardiac injury ranging from 2 to 15 ng/ml and measured these proteins across three time points in six patients undergoing alcohol septal ablation for hypertrophic obstructive cardiomyopathy 117. Addona and coworkers used a combination of discovery and targeted proteomics in the context of planned myocardial infarction (PMI) for treatment of hypertrophic cardiomyopathy and myocardial infarction (MI) 118.…”

A candidate liquid chromatography mass spectrometry reference method for the quantification of the cardiac marker 1-32 B-type natriuretic peptide

“…Over the past decade a number of papers have been published on the development of methods for the quantification of BNP by mass spectrometry (MS) [10,[12][13][14][15][16]. Issues including the very low concentration of BNP in plasma, the difficulties associated with measuring large intact peptides by MS and poor stability of BNP in plasma (half-life of 22.6 min) have however limited their further development for application in clinical laboratories.…”