Protein Protocols Protein Protocols

Reverse-Phase HPLC Separation 533
78
Reverse-Phase HPLC Separation of Enzymatic Digests
of Proteins
Kathryn L. Stone and Kenneth R. Williams
1. Introduction
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 reversephase
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, 8) and
automated Edman sequencing.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
533