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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.

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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.

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  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

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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

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