ABSTRACT
In addition to crucial roles in normal human biology, peptide metabolites of the renin-angiotensin (RAS) and kallikrein-kinin systems (KKS) have been reported to be altered in COVID-19 patients. Here, we evaluate new data on RAS and KKS peptides in COVID-19 patient serum obtained from a recently developed, fully validated, and optimized stable isotope labeling LC-MS peptide assay. We found that the RAS peptides angiotensin (ANG) 1, 2, 1-5, and 1-7 were downregulated compared to COVID-free surrogate controls, while the KKS peptides Brad, Brad 1-8, and Brad 1-7 were upregulated. This paper focuses on uncovering the possible diagnostic value of these peptides using receiver operating characteristic (ROC) analyses of these data. ROC plots confirmed that all of the analyte peptides in 80 serum samples from COVID-19 patients were significantly altered from "normal" values of the control samples. The best diagnostic sensitivities and selectivities for COVID vs no COVID were found in ROC plots for Brad and Brad 1-7 (both 99% sensitivity, 100% selectivity). We then analyzed levels of all the peptides grouped according to preassigned values of the World Health Organization (WHO) COVID-19 Severity Index. ROC plots differentiated patients with a high WHO severity index from those with a low WHO severity index with moderate success, with BRAD (73% sensitivity, 79% selectivity) and Ang 1-7 (75% sensitivity, 65% selectivity) giving the best diagnostic performance. Results suggest the possible diagnostic value of these peptides as biomarkers to help identify moderate and serious COVID-19 cases at relatively early stages.
ABSTRACT
Angiotensin and kinin metabolic pathways are reported to be altered by many diseases, including COVID-19. Monitoring levels of these peptide metabolites is important for understanding mechanisms of disease processes. In this paper, we report dimethyl labeling of amines in peptides by addition of formaldehyde to samples and deutero-formaldehyde to internal standards to generate nearly identical isotopic standards with 4 m/z units larger per amine group than the corresponding analyte. We apply this approach to rapid, multiplexed, absolute LC-MS/MS quantitation of renin angiotensin system (RAS) and kallikrein-kinin system (KKS) peptides in human blood serum. Limits of detection (LODs) were obtained in the low pg mL-1 range with 3 orders of magnitude dynamic ranges, appropriate for determinations of normal and elevated levels of the target peptides in blood serum and plasma. Accuracy is within ±15% at concentrations above the limit of quantitation, as validated by spike-recovery in serum samples. Applicability was demonstrated by measuring RAS and KKS peptides in serum from COVID-19 patients, but is extendable to any class of peptides or other small molecules bearing reactive -NH2 groups.
