Peptide Analysis by HPLC
Karen M. Gooding
Eli Lilly and Company, Indianapolis, Indiana, U.S.A.
Introduction
The rapid advancement in peptide research over the past 25 years must be attributed, in part, to the effectiveness of high-performance liquid chromatography (HPLC), particularly reversed-phase chromatography, in the separation and analysis of peptides. The resolution and selectivity of this technique allows peptides to be effectively isolated and purified from closely related substances. It also separates most or all of the components of complex biological mixtures such as tryptic digests of proteins.
Modes of HPLC
Peptides, which are composed of amino acids linked by amide bonds, are often found in random coil to semi-defined conformations, depending on their lengths and structures. As such, most of the composite amino acids are available to interact with the bonded phase of an HPLC support. Although the variety of amino acid characteristics, such as charge, polarity, and hydropho-bicity, would suggest that multiple modes of HPLC would be effective for separation, reversed-phase chro-matography has shown the ultimate success in selectivity and resolution of peptides. Hydrophilic interaction chromatography is a good alternative for hydrophilic peptides or other mixtures that are not separated well by reversed-phase chromatography, whereas cation-exchange chromatography can be effective for highly cationic species. Size-exclusion chromatography is a difficult technique for analysis of peptides, due to their varying solubilities and high degrees of hydrophobicity and charge; however, it is invaluable for resolution from dimers and aggregates.
Reversed-Phase Chromatography
Reversed-phase chromatography (RPC) is a method in which molecules are bound hydrophobically to nonpolar ligands in the presence of a polar solvent. Solutes are generally bound in an acidic mobile phase with elu-tion occurring during a gradient to an organic solvent.
Molecules will tend to be unfolded due to the combination of acidic and organic mobile phases and the hy-drophobic bonded phase. Consequently, binding involves most of the amino acids, depending on the conformation. RPC can differentiate peptides which vary in the positions of their amino acids, as well as in their identities, thus making it a powerful analysis tool. Very high selectivity is attained even for complex biological mixtures, such as the tryptic digest of human growth hormone shown in Fig. 1.
There are many ligands used for RPC, but the most popular for peptide analysis are octadecyl (C18) and octyl (C8) chains. Little difference in selectivity for peptides is observed with ligand chain-length variation; however, mass recovery of very hydrophobic species may be enhanced on the shorter chains. Many
Fig. 1 Tryptic maps of the intact and (insets) degraded forms of recombinant human growth hormone (RHGH). The separation was achieved on a Nucleosil C-18 column (150 X 3.9 mm inner diameter) with a 120-min mobile-phase gradient from 10 mM potassium phosphate (pH 2.85) to 60% acetonitrile in the starting buffer. The flow rate was 1 mL/min and detection was at 214 nm. [Reprinted from J. Frenz, W.S. Hancock, W. J. Henzel and Cs. Horvath, in HPLC of Biological Macro-molecules: Methods and Applications (K. M. Gooding and F. E. Regnier, eds.), Marcel Dekker, Inc., New York, 1990, p. 145.]
Fig. 1 Tryptic maps of the intact and (insets) degraded forms of recombinant human growth hormone (RHGH). The separation was achieved on a Nucleosil C-18 column (150 X 3.9 mm inner diameter) with a 120-min mobile-phase gradient from 10 mM potassium phosphate (pH 2.85) to 60% acetonitrile in the starting buffer. The flow rate was 1 mL/min and detection was at 214 nm. [Reprinted from J. Frenz, W.S. Hancock, W. J. Henzel and Cs. Horvath, in HPLC of Biological Macro-molecules: Methods and Applications (K. M. Gooding and F. E. Regnier, eds.), Marcel Dekker, Inc., New York, 1990, p. 145.]
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