Reproducibility and Variability of Protein Analytes Measured Using a Multiplexed Modified Aptamer Assay

There is growing interest in the use of multiplexed aptamer-based assays for large-scale proteomic studies. However, the analytic, short- and long-term variation of the measured proteins is largely uncharacterized.

We quantified 4001 plasma protein analytes from 42 participants in the Atherosclerosis Risk in Communities (ARIC) Study in split samples and at multiple visits using a multiplexed modified aptamer assay. We calculated the CV, Spearman correlation, and intraclass correlation (ICC) between split samples and evaluated the short-term (4-9 weeks) and long-term (approximately 20 years) variability using paired t-tests with log-transformed protein concentrations and Bonferroni-corrected significance thresholds. We performed principal component (PC) analysis of protein analyte concentrations and evaluated their associations with age, sex, race, and estimated glomerular filtration rate (eGFR).

The mean baseline age was 57 years at the first visit, 43% of participants were male and 57% were white. Among 3693 protein analytes that passed quality control, half (n = 1846) had CVs < 5.0%, Spearman correlations > 0.89, and ICCs > 0.96 among the split samples. Over the short term, only 1 analyte had a statistically significant difference between the 2 time points, whereas, over approximately 20 years, 866 analytes (23.4%) had statistically significant differences (P < 1.4 × 10^-5, 681 increased, 185 decreased). PC1 had high correlations with age (-0.73) and eGFR (0.60). PC2 had moderate correlation with male sex (0.18) and white race (0.31).

Multiplexed modified aptamer technology can assay thousands of proteins with excellent precision. Our results support the potential for large-scale studies of the plasma proteome over the lifespan.