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.

Purification of naturally occurring peptides by reversed-phase HPLC

Nature Protocols volume 2, pages 191–197 (2007)Cite this article

Abstract

Reversed-phase high performance liquid chromatography (HPLC) has become the method of choice for the purification of peptides and small proteins (Mr < 10,000 Da) from natural sources. The technique combines high resolution and recovery with ease and speed of operation and is applicable to a wide range of peptides with different physicochemical properties. This protocol describes procedures for (1) the extraction of a biologically active peptide from animal tissue, (2) concentration of the extracts and partial purification on Sep-Pak cartridges, and (3) purification to near homogeneity on a range of silica-based HPLC columns. Standard operating procedures involve acetonitrile as organic modifier, trifluoroacetic acid as ion-pairing reagent and sequential chromatographies on octadecyl (C18), butyl (C4) and diphenyl wide-pore (300 Å) columns under gradient elution conditions. The limiting factor in the time taken to isolate a peptide is usually the speed at which assays to detect the peptide can be performed, but purifications can generally be accomplished within 1 or 2 weeks.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

Buy

All prices are NET prices.

Figure 1
Figure 2: Purification of NPY from an extract of the whole brain of the caecilian amphibian Typhlonectes natans by sequential chromatography on Vydac (a) C18 semipreparative, (b) C4 analytical, (c) diphenyl analytical and (d) C18 analytical columns.
Figure 3: Final purification steps on an analytical Vydac C18 column in the isolation of endothelin-3 from frog liver17.

References

  1. Jorpes, J.E. & Mutt, V. Contemporary developments in the biochemistry of the gastrointestinal hormones. Recent Prog. Horm. Res. 23, 483–503 (1967).

    PubMed  Google Scholar 

  2. Guillemin, R., Sakiz, E. & Ward, D.N. Further purification of TSH-releasing factor (TRF) from sheep hypothalamic tissues, with observations on the amino acid composition. Proc. Soc. Exp. Biol. Med. 118, 1132–1137 (1965).

    CAS  Article  Google Scholar 

  3. Schally, A.V., Bowers, C.Y., Redding, T.W. & Barrett, J.F. Isolation of thyrotropin releasing factor (TRF) from porcine hypothalamus. Biochem. Biophys. Res. Commun. 25, 165–169 (1966).

    CAS  Article  Google Scholar 

  4. Ebersole, T.J., Conlon, J.M., Goetz, F.W. & Boyd, S.K. Characterization and distribution of neuropeptide Y in the brain of a caecilian amphibian. Peptides 22, 325–334 (2001).

    CAS  Article  Google Scholar 

  5. Gritti, F. & Guiochon, G. Surface heterogeneity of six commercial brands of end-capped C18-bonded silica RPLC separations. Anal. Chem. 75, 5726–5738 (2003).

    CAS  Article  Google Scholar 

  6. Krokhin, O.V. et al. Use of peptide retention time prediction for protein identification by off-line reversed-phase HPLC-MALDI MS/MS. Anal. Chem. 78, 6265–6269 (2006).

    CAS  Article  Google Scholar 

  7. Pearson, J.D. & McCroskey, M.C. Perfluorinated acid alternatives to trifluoroacetic acid for reversed-phase high-performance liquid chromatography. J. Chromatogr. A 746, 277–281 (1996).

    CAS  Article  Google Scholar 

  8. Fexby, S. & Bulow, L. Hydrophobic peptide tags as tools in bioseparation. Trends Biotechnol. 22, 511–516 (2004).

    CAS  Article  Google Scholar 

  9. Mant, C.T., Litowski, J.R. & Hodges, R.S. Hydrophilic interaction/cation-exchange chromatography for separation of amphipathic alpha-helical peptides. J. Chromatogr. A 816, 65–78 (1998).

    CAS  Article  Google Scholar 

  10. Legido-Quigley, C., Marlin, N. & Smith, N.W. Comparison of styrene–divinylbenzene-based monoliths and Vydac nano-liquid chromatography columns for protein analysis. J. Chromatogr. A 1030, 195–200 (2004).

    CAS  Article  Google Scholar 

  11. Haas, R. & Rosenberry, T.L. Protein denaturation by addition and removal of acetonitrile: Application to tryptic digestion of acetylcholinesterase. Anal. Biochem. 224, 425–427 (1995).

    CAS  Article  Google Scholar 

  12. Conlon, J.M. Measurement of neuerokinin B by radioimmunoassay. In Methods in Neurosciences. Vol. 6 (ed. Conn, P.M.) 221–231 (Academic Press, San Diego, California, 1991).

    Google Scholar 

  13. Goraya, J. et al. Peptides with antimicrobial activity from four different families isolated from the skins of the North American frogs, Rana luteiventris, Rana berlandieri and Rana pipiens . Eur. J. Biochem. 267, 894–900 (2000).

    CAS  Article  Google Scholar 

  14. Marley, P.D. & Rehfeld, J.F. Extraction techniques for gastrins and cholecystokinins in the rat central nervous system. J. Neurochem. 42, 1515–1522 (1984).

    CAS  Article  Google Scholar 

  15. Henry, J.S., Lance, V.A. & Conlon, J.M. Purification and characterization of insulin from Przewalski's horse, zebra, rhino and tapir (Perissodactyla). Gen. Comp. Endocrinol. 89, 299–308 (1993).

    CAS  Article  Google Scholar 

  16. Skribanek, Z., Mezo, G., Mak, H. & Hudecz, F. Mass spectrometric and chemical stability of the Asp–Pro bond in herpes simplex virus epitope peptides compared with X–Pro bonds of related sequences. J. Pept. Sci. 8, 398–406 (2002).

    CAS  Article  Google Scholar 

  17. Wang, Y. et al. Structural characterization and effects on corticosteroid secretion of endothelin-1 and endothelin-3 from the frog, Rana ridibunda . J. Mol. Endocrinol. 24, 285–293 (2000).

    Article  Google Scholar 

Download references

Author information

Affiliations

  1. Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Al-Ain, United Arab Emirates

    J Michael Conlon

Authors
  1. J Michael Conlon
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J Michael Conlon.

Ethics declarations

Competing interests

The author declares no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Conlon, J. Purification of naturally occurring peptides by reversed-phase HPLC. Nat Protoc 2, 191–197 (2007). https://doi.org/10.1038/nprot.2006.437

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.437

Further reading

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