In vitro metabolism of synthetic Elabela/Toddler (ELA-32) peptide in human plasma and kidney homogenates analyzed with mass spectrometry and validation of endogenous peptide quantification in tissues by ELISA

ELA is an apelin receptor agonist that was first highlighted for its role in heart development in zebrafish and has since been shown to beneficial in several animal models of cardiovascular disease. ELA is produced in an inactive form, as a preproprotein, and is then cleaved to produced biologically active peptides of various lengths. While the resulting peptide sequences can be predicted based on putative cleavages sites, the actual peptides produced in human tissue have yet to be shown. In a recent paper, Nyimanu et al. analyzed intact peptides harvested from serum and tissue by LC-MS/MS and then performed de novo sequencing to determine the various isoforms that are generated in situ. This data is an important steppingstone to future studies that examine how the different forms of ELA individually impact signaling networks within the human body.

How was PEAKS used?

PEAKS software was used to de novo sequence the isoforms of ELA. Following de novo, flexibility in the analysis parameters allowed the researchers to perform searches against Uniprot as well as custom databases, and to include multiple variable post-translational modifications (PTM) in their analysis. A decoy fusion database was used to accurately estimate the false-discovery rate (FDR), which the authors set at 1%.

Nyimanu, Duuamene, et al. “In Vitro Metabolism of Synthetic Elabela/Toddler (ELA-32) Peptide in Human Plasma and Kidney Homogenates Analyzed with Mass Spectrometry and Validation of Endogenous Peptide Quantification in Tissues by ELISA.” Peptides, Elsevier BV, Nov. 2021, p. 170642. Crossref, doi:10.1016/j.peptides.2021.170642.

Abstract

Background

Elabela/Toddler (ELA) is a novel endogenous ligand of the apelin receptor, whose signalling has emerged as a therapeutic target, for example, in cardiovascular disease and cancer. Shorter forms of ELA-32 have been predicted, including ELA-21 and ELA-11, but metabolism and stability of ELA-32 in humans is poorly understood. We, therefore, developed an LC–MS/MS assay to identify ELA-32 metabolites in human plasma and tissues.

Method

Human kidney homogenates or plasma were incubated at 37 °C with ELA-32 and aliquots withdrawn over 2−4 h into guanidine hydrochloride. Proteins were precipitated and supernatant solid-phase extracted. Peptides were extracted from coronary artery, brain and kidney by immunoprecipitation or solid-phase extraction following acidification. All samples were reduced and alkylated before analysis on an Orbitrap mass spectrometer in high and nano flow mode.

Results

The half-life of ELA-32 in plasma and kidney were 47.2 ± 5.7 min and 44.2 ± 3 s, respectively. Using PEAKS Studio and manual data analysis, the most important fragments of ELA-32 with potential biological activity identified were ELA-11, ELA-16, ELA-19 and ELA-20. The corresponding fragments resulting from the loss of C-terminal amino acids were also identified. Endogenous levels of these peptides could not be measured, as ELA peptides are prone to oxidation and poor chromatographic peaks.

Conclusions

The relatively long ELA plasma half-life observed and identification of a potentially more stable fragment, ELA-16, may suggest that ELA could be a better tool compound and novel template for the development of new drugs acting at the apelin receptor.

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