Reverse-Phase HPLC Separation of Enzymatic Digests of Proteins

  • Kathryn L. Stone
  • Kenneth R. Williams
Part of the Springer Protocols Handbooks book series (SPH)


The ability of reverse-phase HPLC to resolve complex mixtures of peptides within a few hours’ time in a volatile solvent makes it the current method of choice for fractionating enzymatic digests of proteins. In general, we find that peptides that are less than about 30 residues in length usually separate based on their content of hydrophobic amino acids and that their relative elution positions can be reasonably accurately predicted from published retention coefficients (1,2). Since proteins often retain some degree of folding under the conditions used for reverse-phase HPLC, the more relevant parameter in this instance is probably surface rather than total hydrophobicity. Although larger peptides and proteins may be separated on HPLC, sometimes their tight binding, slow kinetics of release, propensity to aggregate, and relative insolubility in the usual acetonitrile/0.05% trifluoroacetic acid mobile phase results in broad peaks and/or carryover to successive chromatograms. In our experience, these problems are seldom seen with peptides that are less than about 30 residues in length, which thus makes reverse-phase HPLC an ideal method for fractionating tryptic and lysyl endopeptidase digests of proteins. Although it is sometimes possible to improve a particular separation by lessening the gradient slope in that region of the chromatogram, generally, enzymatic digests from a wide variety of proteins can be reasonably well fractionated using a single gradient that might extend over 1–2 h. Another advantage of reverse-phase HPLC is its excellent reproducibility which greatly facilitates using comparative HPLC peptide mapping to detect subtle alterations between otherwise identical proteins. Applications of this approach might include identifying point mutations as well as sites of chemical and posttranslational modification and demonstrating precursor/product relationships. Finally, since peptides are isolated from reverse-phase HPLC in aqueous mixtures of acetonitrile and 0.05% TFA, they are ideally suited for subsequent analysis by matrix-assisted laser desorption mass spectrometry (MALDI-MS, see Chapter 91) and automated Edman sequencing.


Gradient Time Peak Delay Linear Flow Velocity Identify Point Mutation Disulfide Interchange 
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Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Kathryn L. Stone
    • 1
  • Kenneth R. Williams
    • 1
  1. 1.Biotechnology Resource LaboratoryYale UniversityNew Haven

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