Same-Sample Profiling of Renin-Angiotensin Peptides and Enzymes in Mouse Brain Biopsies by Serial Extraction and High-Resolution Mass Spectrometry

Abstract The brain renin–angiotensin system (RAS) comprises biologically active peptides generated through enzymatic cascades that regulate cardiovascular and neurobiological processes and are implicated in cardiometabolic and neurodegenerative diseases. However, molecular characterization of brain RAS components remains challenging due to their low abundance and limitations of existing analytical approaches. Conventional mass spectrometry (MS) workflows typically require separate samples for peptidomic and proteomic analyses, increasing sample consumption and biological variability. Here, we developed a same-sample serial extraction approach for paired measurement of angiotensin peptides and RAS enzymes from limited biopsies of mouse cortex and brainstem, regions associated with memory and cardiometabolic regulation. Peptides were first extracted with acidified acetonitrile, and proteins were then recovered from the remaining pellet for bottom-up proteomic analysis. Angiotensin peptides were quantified by capillary electrophoresis (CE)–electrospray ionization (ESI)–parallel reaction monitoring (PRM)-MS, while RAS enzymes were measured by nano-flow liquid chromatography–ESI–PRM-MS with complementary discovery proteomic analysis. The first extraction recovered more than 94% of measurable endogenous angiotensin peptide signal, while protein identification and quantification remained comparable to the conventional separate-sample workflow. This strategy halved animal use. CE–ESI–PRM-MS achieved low-attomole sensitivity, with a lower limit of detection estimated at ∼7 amol for angiotensin III. Applied to mouse cortex and brainstem, the workflow profiled 7 angiotensin peptides and 7 RAS enzymes, revealing a more prominent angiotensin-processing peptide-enzyme profile in brainstem than cortex. This sample-sparing analytical strategy enables matched peptide and protein measurements from the same tissue sample and is broadly applicable to pathway-focused studies in scarce biological material.