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

  • Camille Lombard-Banek
  • Zhe Yu
  • Adam P. Swiercz
  • Paul J. Marvar
  • Peter NemesEmail author
Paper in Forefront
Part of the following topical collections:
  1. Young Investigators in (Bio-)Analytical Chemistry


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.


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


Author contributions

Peter Nemes, Camille Lombard-Banek, and Paul J. Marvar designed the research and interpreted the data. Zhe Yu and Adam P. Swiercz performed the water deprivation treatment and collected the tissues. Camille Lombard-Banek measured the samples. Camille Lombard-Banek and Peter Nemes analyzed the data and interpreted the results. Camille Lombard-Banek, Peter Nemes, and Paul J. Marvar wrote the manuscript. All the coauthors commented on the manuscript.

Funding information

The reported work was partially supported by the Arnold and Mabel Beckman Foundation Beckman Young Investigator Grant (to P.N.) and the National Institutes of Health grants 1R35GM124755 (to P.N.) and R01HL137103 (to P.J.M.).

Compliance with ethical standards

All protocols regarding the humane treatment of animals were approved by the Institutional Animal Care and Use Committee of The George Washington University (IACUC no. A279).

Conflict of interest

The authors declare that they have no conflicts of interest.


  1. 1.
    Vickers C, Hales P, Kaushik V, Dick L, Gavin J, Tang J, et al. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. J Biol Chem. 2002;277(17):14838–43. Scholar
  2. 2.
    Coble JP, Grobe JL, Johnson AK, Sigmund CD. Mechanisms of brain renin angiotensin system-induced drinking and blood pressure: importance of the subfornical organ. Am J Physiol Regul Integr Comp Physiol. 2015;308(4):R238–R49. Scholar
  3. 3.
    Forrester SJ, Booz GW, Sigmund CD, Coffman TM, Kawai T, Rizzo V, et al. Angiotensin II signal transduction: an update on mechanisms of physiology and pathophysiology. Physiol Rev. 2018;98(3):1627–738. Scholar
  4. 4.
    Mazzolai L, Pedrazzini T, Nicoud F, Gabbiani G, Brunner HR, Nussberger J. Increased cardiac angiotensin II levels induce right and left ventricular hypertrophy in normotensive mice. Hypertension. 2000;35(4):985–91. Scholar
  5. 5.
    Weiss D, Kools JJ, Taylor WR. Angiotensin II-induced hypertension accelerates the development of atherosclerosis in ApoE-deficient mice. Circulation. 1999;100(18):474. Scholar
  6. 6.
    Nakagawa P, Sigmund CD. How is the brain renin-angiotensin system regulated? Hypertension. 2017;70(1):10–8. Scholar
  7. 7.
    Uijl E, Ren LW, Danser AHJ. Angiotensin generation in the brain: a re-evaluation. Clin Sci. 2018;132(8):839–50. Scholar
  8. 8.
    Phillips MI. Angiotensin in brain. Neuroendocrinology. 1978;25(6):354–77. Scholar
  9. 9.
    McKinley MJ, Albiston AL, Allen AM, Mathai ML, May CN, McAllen RM, et al. The brain renin-angiotensin system: location and physiological roles. Int J Biochem Cell Biol. 2003;35(6):901–18. Scholar
  10. 10.
    Grobe JL, Xu D, Sigmund CD. An intracellular renin-angiotensin system in neurons: fact, hypothesis, or fantasy. Physiology. 2008;23(4):187–93. Scholar
  11. 11.
    Wright JW, Harding JW. Importance of the brain angiotensin system in Parkinson’s disease. Parkinsons Dis. 2012:860923.
  12. 12.
    von Bohlen und Halbach O. Angiotensin IV in the central nervous system. Cell Tissue Res. 2003;311(1):1–9. Scholar
  13. 13.
    Pyner S. Neurochemistry of the paraventricular nucleus of the hypothalamus: implications for cardiovascular regulation. J Chem Neuroanat. 2009;38(3):197–208. Scholar
  14. 14.
    Jiang F, Yang JM, Zhang YT, Dong M, Wang SX, Zhang Q, et al. Angiotensin-converting enzyme 2 and angiotensin 1-7: novel therapeutic targets. Nat Rev Cardiol. 2014;11(7):413–26. Scholar
  15. 15.
    Yugandhar VG, Clark MA. Angiotensin III: a physiological relevant peptide of the renin angiotensin system. Peptides. 2013;46:26–32. Scholar
  16. 16.
    Ferrario CM, Trask AJ, Jessup JA. Advances in biochemical and functional roles of angiotensin-converting enzyme 2 and angiotensin-(1-7) in regulation of cardiovascular function. Am J Physiol Heart Circ Physiol. 2005;289(6):H2281–H90. Scholar
  17. 17.
    Sigmund CD, Diz DI, Chappell MC. No brain renin-angiotensin system: deja vu all over again? Hypertension. 2017;69(6):1007–10. Scholar
  18. 18.
    Chappell MC, Brosnihan KB, Diz DI, Ferrario CM. Identification of angiotensin-(1-7) in rat brain - evidence of differential processing of angiotensin peptides. J Biol Chem. 1989;264(28):16518–23.Google Scholar
  19. 19.
    Desilva PE, Husain A, Smeby RR, Khairallah PA. Measurement of immunoactive angiotensin peptides in rat tissues - some pitfalls in angiotensin-II analysis. Anal Biochem. 1988;174(1):80–7. Scholar
  20. 20.
    Herrera M, Sparks MA, Alfonso-Pecchio AR, Harrison-Bernard LM, Coffman TM. Response to lack of specificity of commercial antibodies leads to misidentification of angiotensin type-1 receptor protein. Hypertension. 2013;61(4):E32–E. Scholar
  21. 21.
    Chappell MC. Biochemical evaluation of the renin-angiotensin system: the good, bad, and absolute? Am J Physiol Heart Circ Physiol. 2016;310(2):H137–H52. Scholar
  22. 22.
    Svensson M, Skold K, Nilsson A, Falth M, Nydahl K, Svenningsson P, et al. Neuropeptidomics: MS applied to the discovery of novel peptides from the brain. Anal Chem. 2007;79(1):14–21. Scholar
  23. 23.
    Zestos AG, Kennedy RT. Microdialysis coupled with LC-MS/MS for in vivo neurochemical monitoring. AAPS J. 2017;19(5):1284–93. Scholar
  24. 24.
    Scifo E, Calza G, Fuhrmann M, Soliymani R, Baumann M, Lalowski M. Recent advances in applying mass spectrometry and systems biology to determine brain dynamics. Expert Rev Proteomics. 2017;14(6):545–59. Scholar
  25. 25.
    Peterson AC, Russell JD, Bailey DJ, Westphall MS, Coon JJ. Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics. Mol Cell Proteomics. 2012;11(11):1475–88. Scholar
  26. 26.
    Picotti P, Aebersold R. Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions. Nat Methods. 2012;9(6):555–66. Scholar
  27. 27.
    Cui L, Nithipatikom K, Campbell WB. Simultaneous analysis of angiotensin peptides by LC-MS and LC-MS/MS: metabolism by bovine adrenal endothelial cells. Anal Biochem. 2007;369(1):27–33. Scholar
  28. 28.
    Lortie M, Bark S, Blantz R, Hook V. Detecting low-abundance vasoactive peptides in plasma: progress toward absolute quantitation using nano liquid chromatography-mass spectrometry. Anal Biochem. 2009;394(2):164–70. Scholar
  29. 29.
    Domenig O, Schwager C, van Oyen D, Poglitsch M. Ex vivo equilibrium analysis of the renin-angiotensin-system: clinical implications for diagnosis and treatment of hypertension. Hypertension. 2014;64:A479.Google Scholar
  30. 30.
    Olkowicz M, Radulska A, Suraj J, Kij A, Walczak M, Chlopicki S, et al. Development of a sensitive, accurate and robust liquid chromatography/mass spectrometric method for profiling of angiotensin peptides in plasma and its application for atherosclerotic mice. J Chromatogr A. 2015;1393:37–46. Scholar
  31. 31.
    Tan L, Yu ZR, Zhou XM, Xing D, Luo XY, Peng RF, et al. Antibody-free ultra-high performance liquid chromatography/tandem mass spectrometry measurement of angiotensin I and II using magnetic epitope-imprinted polymers. J Chromatogr A. 2015;1411:69–76. Scholar
  32. 32.
    van Thiel BS, Martini AG, te Riet L, Severs D, Uijl E, Garrelds IM, et al. Brain renin-angiotensin system does it exist? Hypertension. 2017;69(6):1136–44. Scholar
  33. 33.
    Pavo N, Goliasch G, Wurm R, Novak J, Strunk G, Gyongyosi M, et al. Low- and high-renin heart failure phenotypes with clinical implications. Clin Chem. 2018;64(3):597–608. Scholar
  34. 34.
    Hermann K, McDonald W, Unger T, Lang RE, Ganten D. Angiotensin biosynthesis and concentrations in brain of normotensive and hypertensive rats. J Physiol Paris. 1984;79(6):471–80.Google Scholar
  35. 35.
    Bourassa EA, Speth RC. Water deprivation increases angiotensin-converting enzyme but not AT(1) receptor expression in brainstem and paraventricular nucleus of the hypothalamus of the rat. Brain Res. 2010;1319:83–91. Scholar
  36. 36.
    Barber TW, Brockway JA, Higgins LS. The density of tissues in and about the head. Acta Neurol Scand. 1970;46(1):85–92.Google Scholar
  37. 37.
    Lapainis T, Sweedler JV. Contributions of capillary electrophoresis to neuroscience. J Chromatogr A. 2008;1184(1–2):144–58. Scholar
  38. 38.
    Wang J, Ma M, Chen R, Li L. Enhanced neuropeptide profiling via capillary electrophoresis off-line coupled with MALDI FTMS. Anal Chem. 2008;80(16):6168–77. Scholar
  39. 39.
    Jiang XY, Chen RB, Wang JH, Metzler A, Tlusty M, Li LJ. Mass spectral charting of neuropeptidomic expression in the stomatogastric ganglion at multiple developmental stages of the lobster Homarus americanus. ACS Chem Neurosci. 2012;3(6):439–50. Scholar
  40. 40.
    Lombard-Banek C, Moody SA, Nemes P. Single-cell mass spectrometry for discovery proteomics: quantifying translational cell heterogeneity in the 16-cell frog (Xenopus) embryo. Angew Chem Int Ed. 2016;55(7):2454–8. Scholar
  41. 41.
    Lombard-Banek C, Reddy S, Moody SA, Nemes P. Label-free quantification of proteins in single embryonic cells with neural fate in the cleavage-stage frog (Xenopus laevis) embryo using capillary electrophoresis electrospray ionization high-resolution mass spectrometry (CE-ESI-HRMS). Mol Cell Proteomics. 2016;15(8):2756–68. Scholar
  42. 42.
    Choi SB, Zamarbide M, Manzini MC, Nemes P. Tapered-tip capillary electrophoresis nano-electrospray ionization mass spectrometry for ultrasensitive proteomics: the mouse cortex. J Am Soc Mass Spectrom. 2017;28(4):597–607. Scholar
  43. 43.
    Onjiko RM, Moody SA, Nemes P. Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo. Proc Natl Acad Sci U S A. 2015;112(21):6545–50. Scholar
  44. 44.
    Onjiko RM, Morris SE, Moody SA, Nemes P. Single-cell mass spectrometry with multi-solvent extraction identifies metabolic differences between left and right blastomeres in the 8-cell frog (Xenopus) embryo. Analyst. 2016;141(12):3648–56.
  45. 45.
    Onjiko RM, Plotnick DO, Moody SA, Nemes P. Metabolic comparison of dorsal versus ventral cells directly in the live 8-cell frog embryo by microprobe single-cell CE-ESI-MS. Anal Methods. 2017;9(34):4964–70. Scholar
  46. 46.
    Onjiko RM, Portero EP, Moody SA, Nemes P. In situ microprobe single-cell capillary electrophoresis mass spectrometry: metabolic reorganization in single differentiating cells in the live vertebrate (Xenopus laevis) embryo. Anal Chem. 2017;89(13):7069–76. Scholar
  47. 47.
    Onjiko RM, Portero EP, Moody SA, Nemes P. Microprobe capillary electrophoresis mass spectrometry for single-cell metabolomics in live frog (Xenopus laevis) embryos. J Vis Exp. 2017;(130):e56956.
  48. 48.
    Lombard-Banek C, Portero EP, Onjiko RM, Nemes P. New-generation mass spectrometry expands the toolbox of cell and developmental biology. Genesis. 2017;55(1–2).
  49. 49.
    Nemes P, Rubakhin SS, Aerts JT, Sweedler JV. Qualitative and quantitative metabolomic investigation of single neurons by capillary electrophoresis electrospray ionization mass spectrometry. Nat Protoc. 2013;8(4):783–99. Scholar
  50. 50.
    Choi SB, Lombard-Banek C, Munoz-Llancao P, Manzini MC, Nemes P. Enhanced peptide detection toward single-neuron proteomics by reversed-phase fractionation capillary electrophoresis mass spectrometry. J Am Soc Mass Spectrom. 2018;29(5):913–22. Scholar
  51. 51.
    Aerts JT, Louis KR, Crandall SR, Govindaiah G, Cox CL, Sweedler JV. Patch clamp electrophysiology and capillary electrophoresis-mass spectrometry metabolomics for single cell characterization. Anal Chem. 2014;86(6):3203–8. Scholar
  52. 52.
    Parkin MC, Wei H, O’Callaghan JP, Kennedy RT. Sample-dependent effects on the neuropeptidome detected in rat brain tissue preparations by capillary liquid chromatography with tandem mass spectrometry. Anal Chem. 2005;77(19):6331–8. Scholar
  53. 53.
    Rubakhin SS, Page JS, Monroe BR, Sweedler JV. Analysis of cellular release using capillary electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry. Electrophoresis. 2001;22(17):3752–8.;2-H
  54. 54.
    Zhang ZC, Jia CX, Li LJ. Neuropeptide analysis with liquid chromatography-capillary electrophoresis-mass spectrometric imaging. J Sep Sci. 2012;35(14):1779–84. Scholar
  55. 55.
    Culman J, Hohle S, Qadri F, Edling O, Blume A, Lebrun C, et al. Angiotensin as neuromodulator/neurotransmitter in central control of body-fluid and electrolyte homeostasis. Clin Exp Hypertens. 1995;17(1–2):281–93. Scholar
  56. 56.
    Reis LC, Saad WA, Camargo LAA, Menani JV, Silveira JEN, Saad WA. Inhibitory effect of DUP-753 on the drinking responses of rats to central administration of noradrenaline and angiotensin II and to dehydration. Braz J Med Biol Res. 1996;29(4):507–10.Google Scholar
  57. 57.
    Abdelaal AE, Mercer PF, Mogenson GJ. Plasma angiotensin II levels and water intake following beta-adrenergic stimulation, hypovolemia, cellular dehydration and water deprivation. Pharmacol Biochem Behav. 1976;4(3):317–21. Scholar
  58. 58.
    Bekkevold CM, Robertson KL, Reinhard MK, Battles AH, Rowland NE. Dehydration parameters and standards for laboratory mice. J Am Assoc Lab Anim Sci. 2013;52(3):233–9.Google Scholar
  59. 59.
    Paxinos G, Franklin KBJ, Franklin KBJ. The mouse brain in stereotaxic coordinates. 2nd ed. San Diego: Academic Press; 2001.Google Scholar
  60. 60.
    Sun LL, Zhu GJ, Zhao YM, Yan XJ, Mou S, Dovichi NJ. Ultrasensitive and fast bottom-up analysis of femtogram amounts of complex proteome digests. Angew Chem Int Ed. 2013;52(51):13661–4. Scholar
  61. 61.
    MacLean B, Tomazela DM, Shulman N, Chambers M, Finney GL, Frewen B, et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 2010;26(7):966–8. Scholar
  62. 62.
    Zhang CX, Thormann W. Head-column field-amplified sample stacking in binary system capillary electrophoresis: a robust approach providing over 1000-fold sensitivity enhancement. Anal Chem. 1996;68(15):2523–32. Scholar
  63. 63.
    Reiher W, Shirras C, Kahnt J, Baumeister S, Isaac RE, Wegener C. Peptidomics and peptide hormone processing in the drosophila midgut. J Proteome Res. 2011;10(4):1881–92. Scholar
  64. 64.
    Jia CX, Lietz CB, Ye H, Hui LM, Yu Q, Yoo S, et al. A multi-scale strategy for discovery of novel endogenous neuropeptides in the crustacean nervous system. J Proteome. 2013;91:1–12. Scholar
  65. 65.
    Thornton SN. Thirst and hydration: physiology and consequences of dysfunction. Physiol Behav. 2010;100(1):15–21. Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Camille Lombard-Banek
    • 1
  • Zhe Yu
    • 2
  • Adam P. Swiercz
    • 2
  • Paul J. Marvar
    • 2
  • Peter Nemes
    • 1
    Email author
  1. 1.Department of Chemistry & BiochemistryUniversity of MarylandCollege ParkUSA
  2. 2.Department of Pharmacology & Physiology, Institute for NeuroscienceThe George Washington UniversityWashingtonUSA

Personalised recommendations