A pipeline that integrates the discovery and verification of plasma protein biomarkers reveals candidate markers for cardiovascular disease

Abstract

We developed a pipeline to integrate the proteomic technologies used from the discovery to the verification stages of plasma biomarker identification and applied it to identify early biomarkers of cardiac injury from the blood of patients undergoing a therapeutic, planned myocardial infarction (PMI) for treatment of hypertrophic cardiomyopathy. Sampling of blood directly from patient hearts before, during and after controlled myocardial injury ensured enrichment for candidate biomarkers and allowed patients to serve as their own biological controls. LC-MS/MS analyses detected 121 highly differentially expressed proteins, including previously credentialed markers of cardiovascular disease and >100 novel candidate biomarkers for myocardial infarction (MI). Accurate inclusion mass screening (AIMS) qualified a subset of the candidates based on highly specific, targeted detection in peripheral plasma, including some markers unlikely to have been identified without this step. Analyses of peripheral plasma from controls and patients with PMI or spontaneous MI by quantitative multiple reaction monitoring mass spectrometry or immunoassays suggest that the candidate biomarkers may be specific to MI. This study demonstrates that modern proteomic technologies, when coherently integrated, can yield novel cardiovascular biomarkers meriting further evaluation in large, heterogeneous cohorts.

Figure 1

A pipeline for biomarker verification and its application to a human model of myocardial injury. Blood samples were collected from the coronary sinus of patients undergoing alcohol septal ablation for hypertrophic cardiomyopathy (planned myocardial infarction or PMI) before ablation (baseline), and at 10 and 60 min after ablation. The coronary sinus samples, comprising a proximal fluid of the heart, were used for discovery proteomics, using abundant protein depletion and extensive fractionation and LC-MS/MS of peptides to generate a prioritized list of biomarker candidates. Peripheral blood was collected from patients undergoing PMI at the same time points and additional time points extending to 24 h after ablation. Blood collected up to 4 h after ablation was used for analytical qualification by AIMS, a process that determines which of the differentially abundant proteins from the discovery experiments are detectable in peripheral blood. Qualified protein biomarker candidates were subsequently quantitatively measured in peripheral blood using immunoassays (when antibodies were available) and SID-MRM-MS when antibody reagents were not available. Figure was adapted from reference . Blue type indicates proximal fluid. Red type indicates peripheral blood.

Figure 2

Venn diagrams summarizing proteins identified in the coronary sinuses of PMI patients. (a–c) The overlap of proteins identified across all three time points in patients 1, 2 and 3, respectively. Proteins were identified with a minimum of two unique peptides per protein and peptide FDR ≤ 1.5%. (d) We identified 1,105 unique proteins in the nine coronary sinus samples analyzed by LC-MS/MS with >70% of the proteins identified in all three patients. Label-free relative quantification of peptides was done to prioritize candidate proteins for subsequent qualification and verification studies. (e) A minimum of a fivefold change in the MS-derived discovery data between baseline and either the 10-min or 60-min time point was required. 121 proteins met these criteria in all three or any two patients combined.

Figure 3

Kinetic analyses of known and putative biomarkers for acute myocardial infarction in PMI patients from discovery proteomics. (a) Known markers, such as CKM, MB, MPO and FABP showed little to no detection at baseline in coronary sinus plasma followed by a >5-fold increase at 10 min and 60 min after ablation in three PMI patients. (b) Eight potentially novel candidate biomarkers from discovery proteomics. These proteins showed no to little detection at baseline in coronary sinus plasma, then increased in MS abundance by at least fivefold at 10 min or 60 min after ablation in all three PMI patients. MRM-MS assays were configured for ACLP1, myosin light chain 3 and four-and-a-half LIM domain protein 1 to quantify these candidates in peripheral plasma of four PMI patients. Antibodies available for ACBP, ANG, MDK, malate dehydrogenase and ACLP1 were used either in ELISAs or western blot analyses to verify these candidates in additional patients (Supplementary Methods).

Figure 4

Verification of novel candidate biomarkers in peripheral blood of PMI patients by targeted, quantitative MS. Multiplexed SID-MRM-MS–based assays were configured for six candidate proteins to precisely quantify their changes in peripheral blood from PMI patients at 10 min, 60 min and 240 min after ablation. Multiple signature peptides derived from each protein were used to quantify protein levels (Supplementary Tables 7 and 8). Measured concentrations for the four novel proteins ranged from 1 ng/ml to ~50 ng/ml across all patients and time points. Error bars indicate s.e.m. concentration measured at each time point (n = 3). Signature peptides are represented by the first four residues. ACLP1, aortic carboxypeptidase-like protein 1; FHL1, four-and-a-half LIM domain protein 1; MYL3, myosin light chain 3; TPM1, tropomyosin 1. Three known markers of cardiovascular disease were also monitored (Supplementary Table 8b).

Figure 5

Verification of candidate biomarkers by western blot analysis and ELISA. (a) Single antibody reagents suitable for western blot analysis were available for MDK, PTN, malate dehydrogenase 1 (MDH1) and aortic carboxypeptidase-like protein 1 (ACLP1). Kinetic analysis of coronary sinus samples from six patients show consistency in the protein changes between the western blot results shown here and the MS-derived temporal trends shown in Figure 3 for the identical proteins. (b) For ANG, ACBP, and C-C motif chemokine 21 (CCL21), sandwiched immunoassays were either constructed (ANG) or commercially available (ACBP and CCL21), and were used to verify protein changes in peripheral plasma from a larger set of PMI patient samples, control samples and spontaneous MI cohorts. In the PMI cohort (b, left) ELISA results confirm significant changes in these candidate biomarkers as early as 10 min after the onset of myocardial injury. In patients with spontaneous MI (b, right) presenting for acute coronary angiography and intervention, significantly higher levels of these proteins were observed as compared to levels in patients who presented to the cardiac catheterization suite with non-acute coronary artery disease (controls, b, center). BL, baseline; NS, not significant; #, P < 0.05; *, P< 0.01.