Key result
Same-sample serial extraction recovers ~94% of angiotensin peptide signal while matching conventional protein quantification.
Why the study?
Molecular characterization of brain renin-angiotensin system components is challenging due to low abundance, and conventional mass spectrometry requires separate samples for peptidomic and proteomic analyses.
A novel same-sample serial extraction strategy enables matched peptide and protein measurements from limited tissue samples, halving animal use while maintaining high sensitivity.
Reduces animal needs for paired brain RAS peptide-enzyme profiling; leaves open human translation and clinical relevance.
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.
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Li et al. (2026) studied this question. Same-sample serial extraction approach vs. Conventional separate-sample workflow was evaluated on Recovery of measurable endogenous angiotensin peptide signal. A same-sample serial extraction approach recovered >94% of endogenous angiotensin peptide signal from mouse brain biopsies while maintaining comparable protein quantification to conventional workflows.
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