High-Sensitivity Mass Spectrometry of the Renin–Angiotensin System: From Regional Biopsies to Spatial Tissue Imaging

Objective: To develop an integrated analytical platform for high-sensitivity biochemical and spatial analysis of the tissue (brain and kidney) renin–angiotensin system (RAS) for quantification of combined enzyme components and key angiotensin peptides. Hypothesis: We hypothesized that distinct brain regions and renal structures exhibit divergent RAS enzyme and peptide activity that can be resolved using high-sensitivity mass spectrometry (MS) workflows applied to regional tissue biopsies and imaging. Methods: Mouse brain punches (approximately 0.024 mm3) were analyzed using a combined liquid chromatography–parallel reaction monitoring (PRM)–MS and capillary electrophoresis (CE)–PRM–MS workflow for quantifying brain RAS enzymes and endogenous angiotensin peptides. In separate experiments, a matrix-assisted laser desorption/ionization (MALDI)–2 imaging protocol was implemented for spatial map angiotensin peptides in kidney tissue sections. Data and Results: LC–PRM–MS detected and quantified 7 key RAS enzymes, CE–PRM–MS improved sensitivity for angiotensin peptides, achieving approximately two-fold higher separation resolution (Full Width at Half Maximum, FWHM 8 s in CE vs. 14 s in LC), ten-fold wider quantitative linear range (3 log-order vs. 2 log-order of concentrations) than LC. Additionally, CE achieved 25-times higher sensitivity than LC. Their limit of detection (LOD) were 9 amol vs. 230 amol; lower limit of quantification (LLOQ) were 30 amol vs. 769 amol. Five of seven targeted RAS enzymes (ACE, AP-A, AP-N, Carb-P, and ACE2) were quantified elevated in the brainstem relative to the cortex by 26.2, 1.5, 1.6, 3.1, and 2.2 times, respectively. Enzyme prolyl oligopeptidase (PO) was reduced in brainstem compared to cortex. Prorenin receptor (PRR) had a comparable level in both regions. Six of seven targeted angiotensin peptides (angiotensin T, I, II, 1-7, III, and IV) had significantly higher concentrations in brainstem vs. cortex, and their fold change were 3.3, 3.8, 2.1, 2.4, 4.4, and 10.8, respectively. Together, these results indicate regional specific quantification of the brain RAS. For spatial tissue RAS analysis, MALDI–2 imaging sensitively detected endogenous angiotensin 1–7 (m/z 899.47 and 921.47) in mouse kidneys, and following a 24-hour water-deprivation protocol, mice exhibited elevated angiotensin 1–7 localized to specific renal tubular versus ductal segments relative to controls. Conclusions: This integrated platform enables high-sensitivity, region-specific quantification and spatial imaging of RAS components from microdissected brain punches to whole renal tissue sections. Together, these biochemical and imaging workflows establish a framework for quantifying and comparing tissue and region-specific RAS activity, enabling future investigations in cardiovascular and neurodegenerative disease models. Funding: This work was supported by the National Institutes of Health–National Institute on Aging (R21AG086859A to Peter Nemes and Paul J. Marvar). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.