MSACL 2023 Abstract

Introduction:
The Renin Angiotensin Aldosterone System (RAAS) is a complex molecular pathway responsible for the regulation of vascular hemodynamics. When dysregulated, the RAAS contributes to cardiovascular pathologies, such as hypertension, which affects ~40-50% of the US population. Angiotensin (Ang) peptides are the primary effector molecules of the classical RAAS and alternative RAAS pathways. Traditional clinical assay technologies, leveraging antibody-based fluorescence or luminescence, show high levels of cross-reactivity due to the sequence homologies of the Ang peptides. Quantification of these vasoactive peptides using an accurate, high throughput method, such as liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), may provide a better understanding of the mechanisms underlying hypertension that standard blood pressure cuff measurements cannot capture.

Aim:
Establish unique mechanistic insights into the etiology of hypertension by developing a high through-put method to accurately quantitate angiotensin peptides from the RAAS pathway.

Methods:
A LC-MS/MS method was developed to measure four (4) Ang peptides (Ang (1-7), Ang (1-9), Ang I, and Ang II) in human serum. Protease inhibitors, RAAS enzyme-specific inhibitors, and isotope labeled standards are added to 150 ul of serum. Endogenous peptides are isolated from human serum using a two-step hydroalcoholic precipitation followed by mixed-mode solid phase extraction in a 96-well format. LC (Thermo Scientific RSLCnano) separations were performed using capillary flow and a reverse phase trap-elute methodological approach (Waters Corporation nanoEase M/Z Peptide BEH C18 Trap Column, 130Å, 5 µm, 300 µm X 50 mm and nanoEase M/Z Peptide CSH C18 Analytical Column, 130Å, 1.7 µm, 300 µm X 50 mm). MS measurements were completed using a triple quadrupole (Thermo Scientific TSQ Quantiva) set to select for specific peptide precursors and fragments, i.e. multiple reaction monitoring. Peptides were quantified relative to stable isotope labeled synthetic peptides.

Results:
Calibration curves spanning a concentration range of 1 – 1000 fmol/mL were established for all peptides. Linear regression analysis established R2 values ≥ 0.98. Estimated method limits of detection (LOD) are at or below 5 fmol/mL for all 4 peptides. For initial assessments, RAAS peptides were quantified in 25 commercial serum samples. Median values (concentration ranges) were as follows: Ang (1-7) 3.7 fmol/mL (LOD – 37.9 fmol/mL), Ang (1-9) 3.8 fmol/mL (LOD – 16.4 fmol/mL), Ang II 82.0 fmol/mL (LOD – 549.6 fmol/mL), Ang I 27.2 fmol/mL (LOD – 324.0 fmol/mL). Ang II always occurred in the highest concentration, except for one sample where Ang I was the most abundant peptide. In most samples, Ang I was about 4 times lower, and Ang (1-9) and Ang (1-7) were about 25 times lower than Ang II. In one sample, Ang I was about 30 times higher, and Ang (1-7) was about 3 times higher than Ang II.

Conclusion:
This mass spectrometry method enables accurate RAAS peptide quantification in human serum samples. Application of this method will lead to the generation of new data that may provide unique, mechanistic insights into hypertension, as well as renal diseases, arteriosclerosis, and other cardiovascular pathologies associated with the RAAS pathway.