Diversity of Neuropeptide Cell-Cell Signaling Molecules Generated by Proteolytic Processing Revealed by Neuropeptidomics Mass Spectrometry

  • Vivian Hook
  • Christopher B. Lietz
  • Sonia Podvin
  • Tomas Cajka
  • Oliver Fiehn
Focus: 29th Sanibel Conference, Peptidomics: Bridging the Gap Between Proteomics and Metabolomics by MS: Account & Perspective


Neuropeptides are short peptides in the range of 3–40 residues that are secreted for cell-cell communication in neuroendocrine systems. In the nervous system, neuropeptides comprise the largest group of neurotransmitters. In the endocrine system, neuropeptides function as peptide hormones to coordinate intercellular signaling among target physiological systems. The diversity of neuropeptide functions is defined by their distinct primary sequences, peptide lengths, proteolytic processing of pro-neuropeptide precursors, and covalent modifications. Global, untargeted neuropeptidomics mass spectrometry is advantageous for defining the structural features of the thousands to tens of thousands of neuropeptides present in biological systems. Defining neuropeptide structures is the basis for defining the proteolytic processing pathways that convert pro-neuropeptides into active peptides. Neuropeptidomics has revealed that processing of pro-neuropeptides occurs at paired basic residues sites, and at non-basic residue sites. Processing results in neuropeptides with known functions and generates novel peptides representing intervening peptide domains flanked by dibasic residue processing sites, identified by neuropeptidomics. While very short peptide products of 2–4 residues are predicted from pro-neuropeptide dibasic processing sites, such peptides have not been readily identified; therefore, it will be logical to utilize metabolomics to identify very short peptides with neuropeptidomics in future studies. Proteolytic processing is accompanied by covalent post-translational modifications (PTMs) of neuropeptides comprising C-terminal amidation, N-terminal pyroglutamate, disulfide bonds, phosphorylation, sulfation, acetylation, glycosylation, and others. Neuropeptidomics can define PTM features of neuropeptides. In summary, neuropeptidomics for untargeted, global analyses of neuropeptides is essential for elucidation of proteases that generate diverse neuropeptides for cell-cell signaling.

Graphical Abstract


Neuropeptides Proteases Mass spectrometry Peptidomics Cathepsin Pro-protein convertase Enkephalin NPY Regulation Neurotransmission Nervous system Endocrine 


Funding Information

This work was supported by grants from the National Institutes of Health (NIH) to V. Hook (R01 NS094597, R01NS094597-S1) and to O. Fiehn (U24DK097154). C. Lietz was supported by NIH T32MH019934 (awarded to Professor Dilip Jeste, Univ. of Calif., San Diego).


  1. 1.
    Kastin, A.J.: Handbook of Biologically Active Peptides. Elsevier, Amsterdam (2006)Google Scholar
  2. 2.
    Siegel, G.J., Albers, R.W., Fisher, S.K., Uhler, M.D.: Basic Neurochemistry, pp. 363–382. Lippincott Williams and Wilkins, Philadelphia (1999)Google Scholar
  3. 3.
    Hook, V., Funkelstein, L., Lu, D., Bark, S., Wegrzyn, J., Hwang, S.R.: Proteases for processing proneuropeptides into peptide neurotransmitters and hormones. Annu. Rev. Pharmacol. Toxicol. 48, 393–423 (2008)CrossRefGoogle Scholar
  4. 4.
    Yin, P., Hou, X., Romanova, E.V., Sweedler, J.V.: Neuropeptidomics: mass spectrometry-based qualitative and quantitative analysis. Methods Mol. Biol. 789, 223–236 (2011)CrossRefGoogle Scholar
  5. 5.
    Ye, H., Wang, J., Tian, Z., Ma, F., Dowell, J.A., Bremer, Q., Lu, G., Baldo, B., Li, L.: Quantitative mass spectrometry reveals food intake-induced neuropeptide level changes in rat brain: functional assessment of selected neuropeptides as feeding regulators. Mol. Cell Proteomics. 16(11):1922–1937 (2017)Google Scholar
  6. 6.
    Nilsson, A., Stroth, N., Zhang, X., Qi, H., Fälth, M., Sköld, K., Hoyer, D., Andrén, P.E., Svenningsson, P.: Neuropeptidomics of mouse hypothalamus after imipramine treatment reveal somatostatin as a potential mediator of antidepressant effects. Neuropharmacology. 62, 347–357 (2012)CrossRefGoogle Scholar
  7. 7.
    Gupta, N., Bark, S.J., Lu, W.D., Taupenot, L., O'Connor, D.T., Pevzner, P., Hook, V.: Mass spectrometry-based neuropeptidomics of secretory vesicles from human adrenal medullary pheochromocytoma reveals novel peptide products of prohormone processing. J. Proteome Res. 9, 5065–5075 (2010)CrossRefGoogle Scholar
  8. 8.
    Hook, V., Bark, S., Gupta, N., Lortie, M., Lu, W.D., Bandeira, N., Funkelstein, L., Wegrzyn, J., O'Connor, D.T., Pevzner, P.: Neuropeptidomic components generated by proteomic functions in secretory vesicles for cell-cell communication. AAPS J. 12, 635–645 (2010)CrossRefGoogle Scholar
  9. 9.
    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. 394, 164–170 (2009)CrossRefGoogle Scholar
  10. 10.
    Fricker, L.D.: Neuropeptidomics to study peptide processing in animal models of obesity. Endocrinology. 148, 4185–4190 (2007)CrossRefGoogle Scholar
  11. 11.
    Fälth, M., Sköld, K., Svensson, M., Nilsson, A., Fenyö, D., Andren, P.E.: Neuropeptidomics strategies for specific and sensitive identification of endogenous peptides. Mol. Cell. Proteomics. 6, 1188–1197 (2007)CrossRefGoogle Scholar
  12. 12.
    Dowell, J.A., Heyden, W.V., Li, L.: Rat neuropeptidomics by LC-MS/MS and MALDI-FTMS: enhanced dissection and extraction techniques coupled with 2D RP-RP HPLC. J. Proteome Res. 5, 3368–3375 (2006)CrossRefGoogle Scholar
  13. 13.
    Inturrisi, C.E.: Clinical pharmacology of opioids for pain. Clin. J. Pain. 18, S3–13 (2002)CrossRefGoogle Scholar
  14. 14.
    Charbogne, P., Kieffer, B.L., Befort, K.: 15 years of genetic approaches in vivo for addiction research: opioid receptor and peptide gene knockout in mouse models of drug abuse. Neuropharmacology. 76 Pt B, 204–217 (2014)CrossRefGoogle Scholar
  15. 15.
    Podvin, S., Yaksh, T., Hook, V.: The emerging role of spinal dynorphin in chronic pain: a therapeutic perspective. Annu. Rev. Pharmacol. Toxicol. 56, 511–533 (2016)CrossRefGoogle Scholar
  16. 16.
    Steiner, R.A., Hohmann, J.G., Holmes, A., Wrenn, C.C., Cadd, G., Juréus, A., Clifton, D.K., Luo, M., Gutshall, M., Ma, S.Y., Mufson, E.J., Crawley, J.N.: Galanin transgenic mice display cognitive and neurochemical deficits characteristic of Alzheimer’s disease. Proc. Natl. Acad. Sci. U. S. A. 98, 4184–4189 (2001)CrossRefGoogle Scholar
  17. 17.
    Counts, S.E., Perez, S.E., Kahl, U., Bartfai, T., Bowser, R.P., Deecher, D.C., Mash, D.C., Crawley, J.N., Mufson, E.J.: Galanin: neurobiologic mechanisms and therapeutic potential for Alzheimer’s disease. CNS Drug Rev. 7, 445–370 (2011)CrossRefGoogle Scholar
  18. 18.
    Degen, L., Matzinger, D., Drewe, J., Beglinger, C.: The effect of cholecystokinin in controlling appetite and food intake in humans. Peptides. 22, 1265–1269 (2001)CrossRefGoogle Scholar
  19. 19.
    Kageyama, H., Takenoya, F., Hirako, S., Wada, N., Kintaka, Y., Inoue, S., Ota, E., Ogawa, T., Shioda, S.: Neuronal circuits involving neuropeptide Y in hypothalamic arcuate nucleus-mediated feeding regulation. Neuropeptides. 46, 285–289 (2012)CrossRefGoogle Scholar
  20. 20.
    Tordoff, M.G.: Calcium: taste, intake, and appetite. Physiol. Rev. 81, 1567–1597 (2001)CrossRefGoogle Scholar
  21. 21.
    Raddant, A.C., Russo, A.F.: Calcitonin gene-related peptide in migraine: intersection of peripheral inflammation and central modulation. Expert Rev. Mol. Med. 13, e36 (2011)CrossRefGoogle Scholar
  22. 22.
    de Avila, E.D., de Molon, R.S., de Godoi Gonçalves, D.A., Camparis, C.M.: Relationship between levels of neuropeptide substance P in periodontal disease and chronic pain: a literature review. J. Investig. Clin. Dent. 5, 91–97 (2014)CrossRefGoogle Scholar
  23. 23.
    Benoliel, R., Eliav, E., Mannes, A.J., Caudle, R.M., Leeman, S., Iadarola, M.J.: Actions of intrathecal diphtheria toxin-substance P fusion protein on models of persistent pain. Pain. 79, 243–253 (1999)CrossRefGoogle Scholar
  24. 24.
    Felig, P., Baxter, J.D., Frohman, L.A.: Endocrinology and Metabolism, 3rd edn. McGraw Hill Inc., New York (1981)Google Scholar
  25. 25.
    Hook, V., Bandeira, N.: Neuropeptidomics mass spectrometry reveals signaling networks generated by distinct protease pathways in human systems. J. Am. Soc. Mass Spectrom. 26, 1970–1980 (2015)CrossRefGoogle Scholar
  26. 26.
    Seidah, N.G., Prat, A.: The biology and therapeutic targeting of the proprotein convertases. Nat. Rev. Drug Discov. 11, 367–383 (2012)CrossRefGoogle Scholar
  27. 27.
    Hwang, S.R., O'Neill, A., Bark, S., Foulon, T., Hook, V.: Secretory vesicle aminopeptidase B related to neuropeptide processing: molecular identification and subcellular localization to enkephalin- and NPY-containing chromaffin granules. J. Neurochem. 100, 1340–1350 (2007)CrossRefGoogle Scholar
  28. 28.
    Lu, W.D., Funkelstein, L., Toneff, T., Reinheckel, T., Peters, C., Hook, V.: Cathepsin H functions as an aminopeptidase in secretory vesicles for production of enkephalin and galanin peptide neurotransmitters. J. Neurochem. 122, 512–552 (2012)CrossRefGoogle Scholar
  29. 29.
    Fricker, L.D.: Carboxypeptidase E. Annu. Rev. Physiol. 50, 309–321 (1988)CrossRefGoogle Scholar
  30. 30.
    Funkelstein, L., Lu, W.D., Koch, B., Mosier, C., Toneff, T., Taupenot, L., O'Connor, D.T., Reinheckel, T., Peters, C., Hook, V.: Human cathepsin V protease participates in production of enkephalin and NPY neuropeptide neurotransmitters. J. Biol. Chem. 2287, 15232–15241 (2012)CrossRefGoogle Scholar
  31. 31.
    Reiser, J., Adair, B., Reinheckel, T.: Specialized roles for cysteine cathepsins in health and disease. J. Clin. Invest. 120, 3421–3431 (2010)CrossRefGoogle Scholar
  32. 32.
    Brömme, D., Li, Z., Barnes, M., Mehler, E.: Human cathepsin V functional expression, tissue distribution, electrostatic surface potential, enzymatic characterization, and chromosomal localization. Biochemistry. 38, 2377–2385 (1999)CrossRefGoogle Scholar
  33. 33.
    Turk, V., Turk, B., Turk, D.: Lysosomal cysteine proteases: facts and opportunities. EMBO J. 2001(20), 4629–4633 (2001)CrossRefGoogle Scholar
  34. 34.
    Zhou, J., Zhang, Y.Y., Li, Q.Y., Cai, Z.H.: Evolutionary history of cathepsin L (L-like) family genes in vertebrates. Int. J. Biol. Sci. 11(9), 1016–1025 (2015 Jul 14)CrossRefGoogle Scholar
  35. 35.
    Sevenich, L., Hagemann, S., Stoeckle, C., Tolosa, E., Peters, C., Reinheckel, T.: Expression of human cathepsin L or human cathepsin V in mouse thymus mediates positive selection of T helper cells in cathepsin L knock-out mice. Biochimie. 92, 1674–1680 (2010)CrossRefGoogle Scholar
  36. 36.
    Hagemann, S., Günther, T., Dennemärker, J., Lohmüller, T., Brömme, D., Schüle, R., Peters, C., Reinheckel, T.: The human cysteine protease cathepsin V can compensate for murine cathepsin L in mouse epidermis and hair follicles. Eur. J. Cell Biol. 83, 775–780 (2004)CrossRefGoogle Scholar
  37. 37.
    Buchberger, A., Yu, Q., Li, L.: Advances in mass spectrometric tools for probing neuropeptides. Annu. Rev. Anal. Chem. 8, 485–509 (2015)CrossRefGoogle Scholar
  38. 38.
    Svensson, M., Sköld, K., Nilsson, A., Fälth, M., Svenningsson, P., Andrén, P.E.: Neuropeptidomics: expanding proteomics downwards. Biochem. Soc. Trans. 35, 588–593 (2007)CrossRefGoogle Scholar
  39. 39.
    Southey, B.R., Amare, A., Zimmerman, T.A., Rodriguez-Zas, S.L., Sweedler, J.V.: NeuroPred: a tool to predict cleavage sites in neuropeptide precursors and provide the masses of the resulting peptides. Nucleic Acids Res. 34(Web Server issue), W267–W272 (2006)CrossRefGoogle Scholar
  40. 40.
    Tegge, A.N., Southey, B.R., Sweedler, J.V., Rodriguez-Zas, S.L.: Comparative analysis of neuropeptide cleavage sites in human, mouse, rat, and cattle. Mamm. Genome. 19(2), 106–120 (2008)CrossRefGoogle Scholar
  41. 41.
    Clynen, E., Liu, F., Husson, S.J., Landuyt, B., Hayakawa, E., Baggerman, G., Wets, G., Schoofs, L.: Bioinformatic approaches to the identification of novel neuropeptide precursors. Methods Mol. Biol. 615, 357–374 (2010)CrossRefGoogle Scholar
  42. 42.
    Wallace, W.E., Ji, W., Tchekhovskoi, D.V., Phinney, K.W., Stein, S.E.: Mass spectral library quality assurance by inter-library comparison. J. American Society for Mass Spectrometry. 28, 733–738 (2017)CrossRefGoogle Scholar
  43. 43.
    Mann, M., Wilm, M.: Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 66, 4390–4399 (1994)CrossRefGoogle Scholar
  44. 44.
    Heki, N., Noto, M., Hosojima, H.: Analysis of thyrotropin releasing hormone (TRH) in serum and urine by mass fragmentography using GC-MS (author’s transl). Nihon Naibunpi Gakkai Zasshi. 53, 690–702 (1977)Google Scholar
  45. 45.
    Dent, C.L., Humby, T., Lewis, K., Plagge, A., Fischer-Colbrie, R., Wilkins, J.F., Wilkinson, L.S., Isles, A.R.: Impulsive choices in mice lacking imprinted Nesp55. Genes Brain Behav. 15, 693–701 (2016)CrossRefGoogle Scholar
  46. 46.
    Eipper, B.A., Mains, R.E.: Peptide alpha-amidation. Annu. Rev. Physiol. 5, 333–344 (1988)CrossRefGoogle Scholar
  47. 47.
    Schilling, S., Wasternack, C., Demuth, H.U.: Glutaminyl cyclases from animals and plants: a case of functionally convergent protein evolution. Biol. Chem. 389, 983–991 (2008)CrossRefGoogle Scholar
  48. 48.
    Rajpal, G., Schuiki, I., Liu, M., Volchuk, A., Arvan, P.: Action of protein disulfide isomerase on proinsulin exit from endoplasmic reticulum of pancreatic β-cells. J. Biol. Chem. 287, 43–47 (2012)CrossRefGoogle Scholar
  49. 49.
    Takahashi, A., Mizusawa, K.: Posttranslational modifications of proopiomelanocortin in vertebrates and their biological significance. Front. Endocrinol. (Lausanne). 4, 143 (2013)Google Scholar
  50. 50.
    Loh, Y.P., Gainer, H.: Evidence that glycosylation of pro-opiocortin and ACTH influences their proteolysis by trypsin and blood proteases. Mol. Cell. Endocrinol. 20, 35–44 (1980)CrossRefGoogle Scholar
  51. 51.
    Dores, R.M., Steveson, T.C., Price, M.L.: A view of the N-acetylation of alpha-melanocyte-stimulating hormone and beta-endorphin from a phylogenetic perspective. Ann. N. Y. Acad. Sci. 680, 161–174 (1993)CrossRefGoogle Scholar
  52. 52.
    Bleakman, A., Bradbury, A.F., Darby, N.J., Maruthainar, K., Smyth, D.G.: Processing reactions in the later stages of hormone activation. Biochimie. 70, 3–10 (1988)CrossRefGoogle Scholar
  53. 53.
    Vishnuvardhan, D., Beinfeld, M.C.: Role of tyrosine sulfation and serine phosphorylation in the processing of procholecystokinin to amidated cholecystokinin and its secretion in transfected AtT-20 cells. Biochemistry. 39(45), 13825–13830 (2000)CrossRefGoogle Scholar
  54. 54.
    Ji, Y., Leymarie, N., Haeussler, D.J., Bachschmid, M.M., Costello, C.E., Lin, C.: Direct detection of S-palmitoylation by mass spectrometry. Anal. Chem. 85, 11952–11959 (2013)CrossRefGoogle Scholar
  55. 55.
    Chen, G., Zhang, Y., Trinidad, J.C., Dann 3rd., C.: Distinguishing sulfotyrosine containing peptides from their phosphotyrosine counterparts using mass spectrometry. J. Am. Soc. Mass Spectrom. (2018).
  56. 56.
    Hersberger, K.E., Håkansson, K.: Characterization of O-sulfopeptides by negative ion mode tandem mass spectrometry: superior performance of negative ion electron capture dissociation. Anal. Chem. 84, 6370–6377 (2012)CrossRefGoogle Scholar
  57. 57.
    Hook, V., Brennand, K.J., Kim, Y., Toneff, T., Funkelstein, L., Lee, K.C., Ziegler, M., Gage, F.H.: Human iPSC neurons display activity-dependent neurotransmitter secretion: aberrant catecholamine levels in schizophrenia neurons. Stem Cell Reports. 3, 531–538 (2014)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.Skaggs School of Pharmacy and Pharmaceutical SciencesUniversity of California San DiegoLa JollaUSA
  2. 2.Department of Neurosciences, School of MedicineUniversity of California San DiegoLa JollaUSA
  3. 3.West Coast Metabolomics Center, UC Davis Genome CenterUniversity of California DavisDavisUSA

Personalised recommendations