Integration of Proteomic-Based Tools for Improved Biomarkers of Myocardial Injury

Abstract

BACKGROUND:Given the mounting evidence in favor of early pharmacologic and catheter-based interventions for patients across the spectrum of acute coronary syndromes, discovering novel diagnostically sensitive and specific biomarkers that provide biochemical proof of early or reversible myocardial injury could have a substantial positive impact on patient care. CONTENT:To address unmet needs in disease biomarkers, investigators have turned to proteomics approaches. We describe advances in proteomics discovery technologies based on liquid chromatographytandem mass spectrometry that facilitate the unbiased analysis of low-abundance blood proteins. We detail the development of emerging techniques to enhance the biomarker verification process, such as accurate inclusion mass screening, stable isotope dilutionmultiple reaction monitoring-mass spectrometry (SID-MRM-MS), and stable isotope standards with capture by antipeptide antibodies, which combines the advantages of specific immunoaffinity enrichment of a target peptide with the structural specificity and quantitative capabilities of SID-MRM-MS. We highlight new assays incorporating these techniques for troponin I, a representative low-abundance cardiac biomarker, and interleukin-33, an emerging novel marker of myocardial stress for which no existing ELISA exists. We demonstrate that troponin I and interleukin-33 peptides have a linear, dynamic range spanning 4 orders of magnitude and limits of detection of approximately 0.5 g/L back-calculated to the protein concentration.

Synthetic Peptide Arrays for Pathway-Level Protein Monitoring by Liquid Chromatography-Tandem Mass Spectrometry

“…We note that the use of heavy isotope-labeled peptides is also helpful in finding the pairing unlabeled peptide species and confirming relative quantification levels (43,51,52). Software for aligning and integrating multiple LC-MS/MS datasets (53-56) could be used to increase quantification accuracy and detection sensitivity further, whereas modest sample prefractionation can be used to enhance detection limits (57).…”

Top Advances in Functional Genomics and Translational Biology for 2009

“…Selective reaction monitoring completely removes the need for antibodies or as in this case, the need for 2 high affinity specific antibodies while allowing one to obtain quantitative data that is comparable with the data (with similar reproducibility and coefficient of variation) obtained by classic methods such as ELISA or radioimmunoassay. 6 Thus, the selective reaction monitoring technique described in this manuscript provides a novel approach for the targeted discovery and verification of biomarkers in cardiovascular disease.…”

Mass Spectrometry-Based Protein Biomarker Discovery and Measurement: Sensitivity is the Greatest Hurdle

“…A more sensitive technology such as protein array or ELISA may therefore detect a candidate biomarker in a control sample considered to be negative by MS. This should not be considered a false positive.Fortunately, advances in nanotechnology, and affinity capture concentration, are providing a completely new approach to increase the sensitivity of MS for biomarker discovery [5][6][7]. Biomarker "harvesting" nanoparticles rapidly concentrate low abundance proteins for MS, MRM, or immunoassay-based analysis.…”

“…An alternative approach to analyte concentration and pre-separation is antibody affinity capture [7]. In this technology, immobilized antibodies, even those of low specificity for the target analyte, are employed to capture and concentrate the analyte prior to introduction into the MS.…”

“…In this technology, immobilized antibodies, even those of low specificity for the target analyte, are employed to capture and concentrate the analyte prior to introduction into the MS. Recently, the sensitivity of immuno-MS has reached the 0.5 ng/ml range when applied to cardiovascular markers [7]. The success of these two different classes of affinity enrichment technologies provides optimism that the sensitivity and pre-analytical variability of MS can be substantially improved.…”