A Primer to Angiotensin Peptide Isolation, Stability, and Analysis by Nano-Liquid Chromatography with Mass Detection.

The renin-angiotensin system (RAS) is an important element of cardiovascular and renal physiology and targeting the RAS by renin inhibitors, angiotensin (Ang) converting enzyme (ACE) inhibitors and Ang II type 1 receptor antagonists is effective in the treatment of hypertension, heart failure, and atherosclerosis. Quantification of Ang peptides is critical to establish the status of the RAS, but it is challenging due to low Ang peptides concentrations (fmol/mL or fmol/g), abundance of interfering substances, post sampling conversions, and difficulties with the specificity of the assay.In this chapter, we describe a new nano-LC/MS-based methodology for comprehensive, specific, sensitive, and accurate quantification of Ang peptides profile in plasma and tissue. We optimized sample pretreatment method (protein removal (acetonitrile precipitation) followed by solid-phase extraction (C18 silica bonded phase)), chromatographic conditions (reversed-phase nanochromatography with preconcentration), and mass detection (multiple reaction monitoring) of nine peptides: Ang-(1-12), Ang I (1-10), Ang-(1-9), Ang II (1-8), [Ala1]-Ang II, Ang III (2-8), Ang IV (3-8), Ang-(1-7), and [Ala1]-Ang-(1-7). Assessment of plasma and cardiac concentrations of Ang peptides in genetically modified atherosclerotic apolipoprotein E/LDL receptor double knockout (ApoE-/-/LDLR-/-) mice vs. wild types revealed changes in renin-angiotensin system consistent with an overactivation of ACE and impairment of ACE2. The method could be easily adopted for high-throughput analysis and for use in clinical applications such as diagnosis of the RAS abnormalities or monitoring of the RAS inhibition-based therapies.