MASS SPECTROMETRY-BASED QUANTIFICATION OF ACTIVE ANGIOTENSINOGEN IN DIFFERENT VERTEBRATE SPECIES

Objective: The renin-angiotensin-system (RAS) is a peptide hormone system responsible for regulating blood pressure and has been a target for many treatments to lower blood pressure. Newer blood pressure lowering medications employ siRNA or antisense oligonucleotides to reduce the transcription or inhibit the RNA translation of angiotensinogen (AGT), respectively. These medications could be used to treat conditions such as pre-eclampsia and resistant hypertension, which cannot be treated with traditional RAS inhibitors. While antibody methods to quantify AGT have widely spread due to the cheap costs and easy handling, there are concerns regarding sensitivity and selectivity. Therefore, a method was developed based on the selectivity and high sensitivity of liquid chromatography coupled with mass spectrometry (LC-MS/MS). Design and method: In the assay active AGT is hydrolyzed by the serin protease renin until a complete turnover of AGT to angiotensin I is achieved. Further degradation of angiotensin I is prevented by adding a mix of inhibitors specific to the downstream RAS enzymes during the incubation and measured by LC-MS/MS analysis. The linearity and reproducibility of the assay are shown by quantifying quality control samples with four different AGT concentrations on three different dates using the final high-throughput assay. Results: Durin the development of the method renin from different species was tested for its ability to hydrolyze angiotensinogen in other vertebrate species. Species-specific renin had to be used for the complete hydrolyzation of AGT to angiotensin I in samples with mice, rat and human AGT. The incubation with other renin species led to an incomplete AGT turnover of approximately 14.5% for the incubation of rat AGT with human renin, approximately 12.8% for the incubation of rat AGT with mouse renin, or angiotensin I levels below the limit of quantification for other combinations. Using the correct renin in the assay makes it possible to distinguish between AGT varieties in transgenic animal models. Conclusions: The generated data can help support the development of new drugs, to evaluate treatment efficiencies as well as support planning of ex vivo and in vivo RAS studies to provide more insight into the renin-angiotensin-system.