A microanalytical capillary electrophoresis mass spectrometry assay for quantifying angiotensin peptides in the brain

Abstract

The renin-angiotensin system (RAS) of the brain produces a series of biologically active angiotensinogen-derived peptides involved in physiological homeostasis and pathophysiology of disease. Despite significant research efforts to date, a comprehensive understanding of brain RAS physiology is lacking. A significant challenge has been the limited set of bioanalytical assays capable of detecting angiotensin (Ang) peptides at physiologically low concentrations (2-15 fmol/g of wet tissue) and sufficient chemical specificity for unambiguous molecular identifications. Additionally, a complex brain anatomy calls for microanalysis of specific tissue regions, thus further taxing sensitivity requirements for identification and quantification in studies of the RAS. To fill this technology gap, we here developed a microanalytical assay by coupling a laboratory-built capillary electrophoresis (CE) nano-electrospray ionization (nano-ESI) platform to a high-resolution mass spectrometer (HRMS). Using parallel reaction monitoring, we demonstrated that this technology achieved confident identification and quantification of the Ang peptides at approx. 5 amol to 300 zmol sensitivity. This microanalytical assay revealed differential Ang peptide profiles between tissues that were micro-sampled from the subfornical organ and the paraventricular nucleus of the hypothalamus, important brain regions involved in thirst and water homeostasis and neuroendocrine regulation to stress. Microanalytical CE-nano-ESI-HRMS extends the analytical toolbox of neuroscience to help better understand the RAS.

Keywords: Angiotensin; Capillary electrophoresis; High-resolution mass spectrometry; Mouse; Nano-liquid chromatography; Parallel reaction monitoring; Paraventricular nucleus; Peptidomics; Renin-angiotensin system; Subfornical organ.

Figure 1.

Detection of angiotensin (Ang) peptides of the (A) renin-angiotensin system using (B) microanalytical high-resolution mass spectrometry (HRMS). Ang peptides are presented in boxes. Enzymes are inscribed in ovals. Peptides chosen for this study are highlighted in orange. The subfornical organ (SFO) and the paraventricular nucleus (PVN) were sampled by micropunch needles in water-deprived (WD) and control (Ctrl) mice (see circles). Ang peptides were microextracted, separated by a laboratory-built microanalytical capillary electrophoresis (CE) instrument, and detected using parallel reaction monitoring (PRM) HRMS. Key: Scale bar, 2 mm.

Figure 2.

Development of a high-resolution mass spectrometry assay for Ang peptides. (A) Parallel reaction monitoring (PRM) selecting Ang peptides in the quadrupole (Q) for fragmentation via higher-energy collisional dissociation (HCD), followed by orbitrap detection of peptide-specific fragments (e.g., b- and y- ions listed). (B) Experimental optimization of fragmentation efficiency as a function of normalized collision energy (NCE) for HCD. Each data point was a separate analysis of the standard peptide mixture (1×10⁻⁴ g/L) using capillary electrophoresis (CE) high-resolution mass spectrometry (HRMS), analyzed in technical triplicate. Key: Error bars, 1 × S.E.M. (C) Data analysis in the Skyline software extracting specific precursor-fragment transitions for integration of under-the-curve peak areas to be used as a proxy for peptide quantification.

Figure 3.

Experimental characterization of analytical performance. (A) Sample reconstitution in higher acid content enhanced sensitivity by improving field-amplified sample stacking. Key: Error bars, 1 × S.E.M; *p < 0.05 (Student’s t-test). (B) The dynamic range of quantification was tested to be linear over a ~3–4 log-order range for the angiotensin standards, extrapolating to a lower limit of quantification at ~1–10 amol (see Table 1).

Figure 4.

Benchmarking microanalytical capillary electrophoresis (CE) against nano-flow liquid chromatography (nanoLC) for high-resolution mass spectrometry (HRMS). Analyzed sample volumes from the same standard angiotensin mixture (100 μg/L) were: 14 nL for CE and 100 nL for nanoLC. (A) Mass-selected ion traces comparing peptide separation during CE and nanoLC, revealing higher separation efficiency and detection sensitivity by electrophoresis. (B) Comparable summed peak areas (SPA) between the technologies suggest ~10-fold higher sensitivity by CE-HRMS. Key: Error bars, 1 × S.E.M; ns, not significant; **p ≤ 0.01 (Student’s t-test).

Figure 5.

Quantitative comparison of Ang peptide amounts between the subfornical organ (SFO, data in black) and the paraventricular nucleus (PVN, data in grey) in control (Ctrl) and upon water deprivation (WD) of N = 3 male mice. Key: Boxes, 1 × S.E.M.; whiskers, 10–90 percentiles; mid-line, mean.