Aptamer-Based Proteomic Profiling Reveals Novel Candidate Biomarkers and Pathways in Cardiovascular Disease
Background—Single-stranded DNA aptamers are oligonucleotides of approximately 50 base pairs in length selected for their ability to bind proteins with high specificity and affinity. Emerging DNA aptamer-based technologies may address limitations of existing proteomic techniques, including low sample throughput, which have hindered proteomic analyses of large cohorts.
Methods—To identify early biomarkers of myocardial injury, we applied an aptamer-based proteomic platform that measures 1129 proteins to a clinically-relevant perturbational model of "planned" myocardial injury (PMI), patients undergoing septal ablation for hypertrophic cardiomyopathy. Blood samples obtained before, at 10 and 60 minutes after PMI, and protein changes were assessed by repeated measures ANOVA. The generalizability of our PMI findings was evaluated in a spontaneous MI (SMI) cohort (Wilcoxon rank-sum). We then tested the platform's ability to detect associations between proteins and Framingham Risk Score (FRS) components in the Framingham Heart Study (FHS); performing regression analyses for each protein versus each clinical trait.
Results—We found 217 proteins that significantly changed in the peripheral vein blood after PMI in a derivation cohort (n=15; P < 5.70E-5). Seventy-nine of these proteins were validated in an independent PMI cohort (n=15; P < 2.30E-4); > 85% were directionally consistent and reached nominal significance. We detected many protein changes that are novel in the context of myocardial injury, including Dickkopf related protein 4, a WNT pathway inhibitor (peak increase 124%, P = 1.29E-15) and cripto, a growth factor important in cardiac development (peak increase 64%, P = 1.74E-4). Among the 40 validated proteins that increased within 1 hour after PMI, 23 were also elevated in patients with SMI (n=46; P < 0.05). Our FHS studies revealed 156 significant protein associations with the FRS (n=899), including aminoacylase 1 (β = 0.3386, P = 2.54E-22) and trigger factor 2 (β = 0.2846, P = 5.71E-17). Further, we developed a novel workflow integrating DNA-based immunoaffinity with mass spectrometry to analytically validate aptamer specificity.
Conclusions—Our results highlight an emerging proteomics tool capable of profiling over one thousand low abundance analytes with high sensitivity and high precision, applicable both to well-phenotyped perturbational studies as well as large human cohorts.
- Received February 3, 2016.
- Revision received June 14, 2016.
- Accepted June 20, 2016.