Microanalytical Mass Spectrometry Detects RAS Metabolites in Small Identified Mouse Brain Samples

Background There is increasing evidence for cardiovascular disease (CVD) development in post‐traumatic stress disorder (PTSD). The renin‐angiotensin system (RAS) critical for cardiovascular homeostasis, has been identified as potential therapeutic target and link to comorbid PTSD‐CVD development. However, the mechanisms are unclear. To better understand how the RAS contributes to CVD risk in PTSD, measurements of the RAS metabolites, the angiotensin peptides, in targeted brain areas are necessary. The low concentrations of angiotensin peptides in the brain however, challenges modern analytical approaches. This challenge is further compounded when studying small identified areas such as brain tissue punches. As a response to this challenge, we developed a microanalytical mass spectrometry approach to enable the detection of angiotensin peptides from brain tissue punches. Methods Using 6 angiotensin‐like peptide standards, we reconfigured a laboratory‐built capillary electrophoresis electrospray ionization platform (CE‐ESI) platform and a high‐resolution mass spectrometer (HRMS) for targeted peptide detection, essentially serving as a highly confident peptide assay. We benchmarked our approach to nano‐liquid chromatography (LC) ESI‐HRMS, the traditional technology for peptide analysis in HRMS. Results We first developed a targeted approach on a quadrupole‐orbitrap mass spectrometer for the detection and quantification of the 6 selected angiotensin‐like peptides. Comparison of our CE‐ESI‐HRMS approach to the traditional nanoLC‐ESI‐HRMS revealed comparable peptide signals despite a 5‐fold reduction in sample loading. This demonstrated that our platform was suited for the measurement of angiotensin peptides from small sample volumes. The lower‐limit of quantification was established in the pM–nM range for all the selected RAS metabolites. After revising the extraction method to enhance peptide recovery, we applied our methodology to measure different levels of RAS metabolites in brain punches from the subfornical organ (SFO) and the paraventricular nucleus of the hypothalamus (PVN) of control and water deprived (WD) mice. Differences in the level of all RAS metabolites in the SFO concurred with the effect of WD in increasing angiotensin levels. On the other hand, levels for most of the RAS metabolites in the PVN did not change noticeably. Conclusions These results demonstrate that our CE‐ESI‐HRMS based analytical approach enabled the sensitive detection of RAS metabolites in small brain tissue samples, raising a potential to help study the chemistry of the brain RAS during PTSD and CVD. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .