Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Analysis of the proteolysis of bioactive peptides using a peptidomics approach

Nature Protocols volume 8, pages 1730–1742 (2013)Cite this article

Subjects

Abstract

Identifying the peptidases that inactivate bioactive peptides (e.g., peptide hormones and neuropeptides) in mammals is an important unmet challenge. This protocol describes a recent approach that uses liquid chromatography-mass spectrometry (LC-MS) peptidomics to identify endogenous cleavage sites of a bioactive peptide; it also addresses the subsequent biochemical purification of a candidate peptidase on the basis of these cleavage sites and the validation of the candidate peptidase's role in the physiological regulation of the bioactive peptide by examining a peptidase-knockout mouse. We highlight the successful application of this protocol in the discovery that insulin-degrading enzyme (IDE) regulates physiological calcitonin gene–related peptide (CGRP) levels, and we detail the key stages and steps in this approach. This protocol requires 7 d of work; however, the total time for this protocol is highly variable because of its dependence on the availability of biological reagents such as purified enzymes and knockout mice. The protocol is valuable because it expedites the characterization of mammalian peptidases, such as IDE, which in certain instances can be used to develop novel therapeutics.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The protocol is divided into three key stages with a number of individual steps making up each stage.
Figure 2: Lysate peptidomics.
Figure 3: In vivo processing of a bioactive peptide.
Figure 4: Identification of IDE as a candidate CGRP-degrading enzyme.
Figure 5: Overall steps of the RAPID method for IDMS studies.
Figure 6: Bioactive peptide degradation in mouse spinal cord and plasma.

Similar content being viewed by others

References

  1. Bliss, M. The Discovery of Insulin (University of Chicago Press, 2013).

  2. Kimball, C. & Murlin, J.R. Aqueous extracts of pancreas III. Some precipitation reactions of insulin. J. Biol. Chem. 58, 337–346 (1923).

    CAS  Google Scholar 

  3. Bromer, W., Sinn, L. & Behrens, O.K. The amino acid sequence of glucagon. V. Location of amide groups, acid degradation studies and summary of sequential evidence. J. Am. Chem. Soc. 79, 2807–2810 (1957).

    Article  CAS  Google Scholar 

  4. Kastin, A. Handbook of Biologically Active Peptides (Academic Press, 2013).

  5. Itakura, K. et al. Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science 198, 1056–1063 (1977).

    Article  CAS  PubMed  Google Scholar 

  6. Goeddel, D.V. et al. Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc. Natl. Acad. Sci. 76, 106–110 (1979).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Tatemoto, K., Carlquist, M. & Mutt, V. Neuropeptide Y—a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 296, 659–660 (1982).

    Article  CAS  PubMed  Google Scholar 

  8. Said, S.I. & Mutt, V. Polypeptide with broad biological activity: isolation from small intestine. Science 169, 1217–1218 (1970).

    Article  CAS  PubMed  Google Scholar 

  9. Tatemoto, K., Rökaeus, ÅÅ, Jörnvall, H., McDonald, T.J. & Mutt, V. Galanin—a novel biologically active peptide from porcine intestine. FEBS Lett. 164, 124–128 (1983).

    Article  CAS  PubMed  Google Scholar 

  10. Strand, F.L. Neuropeptides (Wiley Online Library, 1999).

  11. Steiner, D.F. The proprotein convertases. Curr. Opin. Chem. Biol. 2, 31–39 (1998).

    Article  CAS  PubMed  Google Scholar 

  12. Marguet, D. et al. Enhanced insulin secretion and improved glucose tolerance in mice lacking CD26. Proc. Natl. Acad. Sci. 97, 6874–6879 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gradman, A.H. et al. Aliskiren, a novel orally effective renin inhibitor, provides dose-dependent antihypertensive efficacy and placebo-like tolerability in hypertensive patients. Circulation 111, 1012–1018 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Patchett, A.A. et al. A new class of angiotensin-converting enzyme inhibitors. Nature 288, 280–283 (1980).

    Article  CAS  PubMed  Google Scholar 

  15. Deacon, C.F. et al. Both subcutaneously and intravenously administered glucagon-like peptide I are rapidly degraded from the NH2-terminus in type II diabetic patients and in healthy subjects. Diabetes 44, 1126–1131 (1995).

    Article  CAS  PubMed  Google Scholar 

  16. Xu, H. & Shields, D. Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells. J. Cell Biol. 122, 1169–1184 (1993).

    Article  CAS  PubMed  Google Scholar 

  17. Jung, L.J. & Scheller, R.H. Peptide processing and targeting in the neuronal secretory pathway. Science 251, 1330–1335 (1991).

    Article  CAS  PubMed  Google Scholar 

  18. Scamuffa, N., Calvo, F., Chretien, M., Seidah, N.G. & Khatib, A.M. Proprotein convertases: lessons from knockouts. FASEB J. 20, 1954–1963 (2006).

    Article  CAS  PubMed  Google Scholar 

  19. Furuta, M. et al. Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2. Proc. Natl. Acad. Sci. 94, 6646–6651 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Schalekamp, M.A., Derkx, F.H. & van den Meiracker, A.H. Renin inhibitors, angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists: relationships between blood pressure responses and effects on the renin-angiotensin system. J. Hypertens. Suppl. 10, S157–S164 (1992).

    Article  CAS  PubMed  Google Scholar 

  21. Kjems, L.L., Holst, J.J., Volund, A. & Madsbad, S. The influence of GLP-1 on glucose-stimulated insulin secretion: effects on beta cell sensitivity in type 2 and nondiabetic subjects. Diabetes 52, 380–386 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Rachman, J., Barrow, B., Levy, J. & Turner, R. Near-normalisation of diurnal glucose concentrations by continuous administration of glucagon-like peptide-1 (GLP-1) in subjects with NIDDM. Diabetologia 40, 205–211 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Rosenblum, J.S. & Kozarich, J.W. Prolyl peptidases: a serine protease subfamily with high potential for drug discovery. Curr. Opin. Chem. Biol. 7, 496–504 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Overall, C.M. & Blobel, C.P. In search of partners: linking extracellular proteases to substrates. Nat. Rev. Mol. Cell Biol. 8, 245–257 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Thornberry, N.A. & Weber, A.E. Discovery of JANUVIA (sitagliptin), a selective dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. Curr. Top. Med. Chem. 7, 557–568 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Augeri, D.J. et al. Discovery and preclinical profile of saxagliptin (BMS-477118): a highly potent, long-acting, orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. J. Med. Chem. 48, 5025–5037 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Toide, K., Okamiya, K., Iwamoto, Y. & Kato, T. Effect of a novel prolyl endopeptidase inhibitor, JTP-4819, on prolyl endopeptidase activity and substance P- and arginine-vasopressin-like immunoreactivity in the brains of aged rats. J. Neurochem. 65, 234–240 (1995).

    Article  CAS  PubMed  Google Scholar 

  28. Tatemoto, K. & Mutt, V. Isolation and characterization of the intestinal peptide porcine PHI (PHI-27), a new member of the glucagon–secretin family. Proc. Natl. Acad. Sci. USA 78, 6603–6607 (1981).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Szecowka, J., Tatemoto, K., Mutt, V. & Efendic, S. Interaction of a newly isolated intestinal polypeptide (PHI) with glucose and arginine to effect the secretion of insulin and glucagon. Life Sci. 26, 435–438 (1980).

    Article  CAS  PubMed  Google Scholar 

  30. Szecowka, J., Lins, P.E., Tatemoto, K. & Efendic, S. Effects of porcine intestinal heptacosapeptide and vasoactive intestinal polypeptide on insulin and glucagon secretion in rats. Endocrinology 112, 1469–1473 (1983).

    Article  CAS  PubMed  Google Scholar 

  31. Rosenfeld, M.G., Amara, S.G. & Evans, R.M. Alternative RNA processing: determining neuronal phenotype. Science 225, 1315–1320 (1984).

    Article  CAS  PubMed  Google Scholar 

  32. Amara, S., Arriza, J., Leff, S. & Swanson, L. Expression in brain of a messenger RNA encoding a novel neuropeptide homologous to calcitonin gene-related peptide. Science 229, 1094–1097 (1985).

    Article  CAS  PubMed  Google Scholar 

  33. Brain, S., Williams, T., Tippins, J. & Morris, H. Calcitonin gene-related peptide is a potent vasodilator. Nature 313, 54–56 (1985).

    Article  CAS  PubMed  Google Scholar 

  34. Ashina, M., Bendtsen, L., Jensen, R., Schifter, S. & Olesen, J. Evidence for increased plasma levels of calcitonin gene-related peptide in migraine outside of attacks. Pain 86, 133–138 (2000).

    Article  CAS  PubMed  Google Scholar 

  35. Ho, T.W. et al. Randomized controlled trial of an oral CGRP receptor antagonist, MK-0974, in acute treatment of migraine. Neurology 70, 1304–1312 (2008).

    Article  CAS  PubMed  Google Scholar 

  36. Lassen, L.H. et al. CGRP may play a causative role in migraine. Cephalalgia 22, 54–61 (2002).

    Article  CAS  PubMed  Google Scholar 

  37. Paone, D.V. et al. Potent, orally bioavailable calcitonin gene-related peptide receptor antagonists for the treatment of migraine: discovery of N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1- (2,2,2-trifluoroethyl)azepan-3-yl]-4- (2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin- 1-yl)piperidine-1-carboxamide (MK-0974). J. Med. Chem. 50, 5564–5567 (2007).

    Article  CAS  PubMed  Google Scholar 

  38. Salvatore, C.A. et al. Pharmacological characterization of MK-0974 [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide], a potent and orally active calcitonin gene-related peptide receptor antagonist for the treatment of migraine. J. Pharmacol. Exp. Therap. 324, 416–421 (2007).

    Article  CAS  Google Scholar 

  39. Nolte, W.M., Tagore, D.M., Lane, W.S. & Saghatelian, A. Peptidomics of prolyl endopeptidase in the central nervous system. Biochemistry 48, 11971–11981 (2009).

    Article  CAS  PubMed  Google Scholar 

  40. Eng, J.K., McCormack, A.L. & Yates, J.R. III. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrometry 5, 976–989 (1994).

    Article  CAS  Google Scholar 

  41. Kim, Y.G., Lone, A.M., Nolte, W.M. & Saghatelian, A. Peptidomics approach to elucidate the proteolytic regulation of bioactive peptides. Proc. Natl. Acad. Sci. USA 109, 8523–8527 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Perkins, D.N., Pappin, D.J., Creasy, D.M. & Cottrell, J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567 (1999).

    Article  CAS  PubMed  Google Scholar 

  43. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  PubMed  Google Scholar 

  44. Gibson, S.J. et al. Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species. J. Neurosci. 4, 3101–3111 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Le Grevès, P., Andersson, K. & Silberring, J. Isolation and identification of CGRP C-terminal fragments in the rat spinal cord. Neuropeptides 31, 19–23 (1997).

    Article  PubMed  Google Scholar 

  46. Katayama, M. et al. Catabolism of calcitonin gene-related peptide and substance P by neutral endopeptidase. Peptides 12, 563–567 (1991).

    Article  CAS  PubMed  Google Scholar 

  47. Le Greves, P., Nyberg, F., Hokfelt, T. & Terenius, L. Calcitonin gene-related peptide is metabolized by an endopeptidase hydrolyzing substance P. Regul. Pept. 25, 277–286 (1989).

    Article  CAS  PubMed  Google Scholar 

  48. Tam, E.K. & Caughey, G.H. Degradation of airway neuropeptides by human lung tryptase. Am. J. Resp. Cell Mol. Biol. 3, 27–32 (1990).

    Article  CAS  Google Scholar 

  49. Tinoco, A.D. et al. A peptidomics strategy to elucidate the proteolytic pathways that inactivate peptide hormones. Biochemistry 50, 2213–2222 (2011).

    Article  CAS  PubMed  Google Scholar 

  50. Anderson, L. & Hunter, C.L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol. Cell Proteomics 5, 573–588 (2006).

    Article  CAS  PubMed  Google Scholar 

  51. Duckworth, W.C., Heinemann, M.A. & Kitabchi, A.E. Purification of insulin-specific protease by affinity chromatography. Proc. Natl. Acad. Sci. USA 69, 3698–3702 (1972).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Ray, K., Hines, C.S. & Rodgers, D.W. Mapping sequence differences between thimet oligopeptidase and neurolysin implicates key residues in substrate recognition. Protein sci. 11, 2237–2246 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kim, Y. & Brophy, E. Rat intestinal brush border membrane peptidases. I. Solubilization, purification, and physicochemical properties of two different forms of the enzyme. J. Biol. Chem. 251, 3199–3205 (1976).

    CAS  PubMed  Google Scholar 

  54. Alvarez-Manilla, G. et al. Tools for glycoproteomic analysis: size exclusion chromatography facilitates identification of tryptic glycopeptides with N-linked glycosylation sites. J. Proteome Res. 5, 701–708 (2006).

    Article  CAS  PubMed  Google Scholar 

  55. Villen, J. & Gygi, S.P. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat. Protoc. 3, 1630–1638 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  56. Slebos, R.J. et al. Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry. J. Proteome Res. 7, 5286–5294 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Buckley, S.J., Collins, P.J. & O′Connor, B.F. The purification and characterisation of novel dipeptidyl peptidase IV-like activity from bovine serum. Int. J. Biochem. Cell Biol. 36, 1281–1296 (2004).

    Article  CAS  PubMed  Google Scholar 

  58. Rawlings, N.D., Barrett, A.J. & Bateman, A. MEROPS: the peptidase database. Nucleic Acids Res. 38, D227–D233 (2010).

    Article  CAS  PubMed  Google Scholar 

  59. Rawlings, N.D., Barrett, A.J. & Bateman, A. MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 40, D343–D350 (2012).

    Article  CAS  PubMed  Google Scholar 

  60. Farris, W. et al. Insulin-degrading enzyme regulates the levels of insulin, amyloid β-protein, and the β-amyloid precursor protein intracellular domain in vivo. Proc. Natl. Acad. Sci. USA 100, 4162–4167 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Zheng, T.S. et al. Deficiency in caspase-9 or caspase-3 induces compensatory caspase activation. Nat. Med. 6, 1241–1247 (2000).

    Article  CAS  PubMed  Google Scholar 

  62. Noone, D., Howell, A., Collery, R. & Devine, K.M. YkdA and YvtA, HtrA-like serine proteases in Bacillus subtilis, engage in negative autoregulation and reciprocal cross-regulation of ykdA and yvtA gene expression. J. Bacteriol. 183, 654–663 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Knight, Z.A. & Shokat, K.M. Features of selective kinase inhibitors. Chem. Biol. 12, 621–637 (2005).

    Article  CAS  PubMed  Google Scholar 

  64. Stengel, A. et al. The RAPID method for blood processing yields new insight in plasma concentrations and molecular forms of circulating gut peptides. Endocrinology 150, 5113–5118 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Coin, I., Beyermann, M. & Bienert, M. Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences. Nat. Protoc. 2, 3247–3256 (2007).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by a Forris Jewitt Moore Fellowship sponsored by Amherst College (A.M.L.), a US National Institutes of Health (NIH) training grant (no. GM007598 to A.M.L.), a Searle Scholar Award (A.S.), a Burroughs Wellcome Fund Career Award in the Biomedical Sciences (A.S.), a NIH grant (no. DP2OD002374 to A.S.), a Korea Basic Science Institute grant (no. T33617 to Y.-G.K.) and a Korea Institute of Science and Technology Institutional Program grant (no. 2E23720-13-053 to Y.-G.K.).

Author information

Authors and Affiliations

  1. Department of Chemical Engineering, Soongsil University, Seoul, South Korea

    Yun-Gon Kim

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    Anna Mari Lone & Alan Saghatelian

Authors
  1. Yun-Gon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anna Mari Lone
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alan Saghatelian
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Experimental procedure development and assembly of the manuscript were performed by Y.-G.K., A.M.L. and A.S.

Corresponding author

Correspondence to Alan Saghatelian.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kim, YG., Lone, A. & Saghatelian, A. Analysis of the proteolysis of bioactive peptides using a peptidomics approach. Nat Protoc 8, 1730–1742 (2013). https://doi.org/10.1038/nprot.2013.104

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2013.104

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing