Peptide Retention Time Prediction Research Articles

Prediction of peptide retention times in reversed-phase high-performance liquid chromatography I. Determination of retention coefficients of amino acid residues of model synthetic peptides

We have examined the contribution of individual amino acid residues to peptide retention on reversed-phase (RP) columns by measuring their effect on retention of a model synthetic peptide: Ac-Gly-X-X-(Leu) 3-(Lys) 2-amide, where X is substituted by the 20 amino acids found in proteins. Consistently similar resolution of the 20 peptides on several RP columns enabled the determination of empirical sets of retention coefficients, describing the hydrophobicity of amino acid residues at pH 2.0 and pH 7.0. The much superior resolution and selectivity obtained with acetonitrile, compared to 2-propanol and methanol, confirmed its value as the best organic eluent for most practical purposes. The necessity of using peptides rather than alkylphenones as internal standards for peptide retention prediction is demonstrated and underlined by the predictive accuracy of our coefficients when applied to the resolution of a mixture of five commercially available synthetic peptide standards on several RP columns. Rules for retention time prediction for linear elution gradients, employing our hydrophobicity parameters, of peptides of known composition are presented and enable the researcher to correct for: (a) instrument variations, (b) varying length or diameter of RP columns, (c) varying n-alkyl chain length and ligand density of RP packings and (d) column aging.

Prediction of peptide retention times in reversed-phases high-performance liquid chromatography during linear gradient elution

The retention behavior of 100 peptides was studied during high-performance liquid chromatography on a C 18 column using aqueous trifluoroacetic acid as the mobile phase and acetonitrile as the mobile phase modifier in a linear gradient elution system. Retention times of the peptides were linearly related to the logarithm of the sum of Rekker's constants (R.F. Rekker, The Hydrophobic Fragmental Constant, Elsevier, Amsterdam, 1977, p. 301) for the constituent amino acid. Assuming this relationship, the best fit constants for this system were computed by non-linear multiple regression analysis. Using the new constants, it is possible to predict retention times for a wide variety of peptides at any slope of linear gradient, if the amino acid composition is known. It also enables accurate prediction of the retention time of peptides, whose amino acid composition in not known, after an analytical run with an alternate gradient.

Prediction of peptide retention times in high-pressure liquid chromatography on the basis of amino acid composition.

Analysis of peptides by reverse-phase high-pressure liquid chromatography would be simplified if retention times could be predicted by summing the contribution to retention of each of the peptide's amino acid side chains. This paper describes the derivation of values ("retention coefficients") that represent the contribution to retention of each of the common amino acids and end groups. Peptide retention times were determined on a Bio-Rad "ODS" column at room temperature with a linear gradient from 0.1 M NaclO(4), pH 7.4 or 2.1, at 0 min to 60% acetonitrile/0.1 M NaclO(4) at 80 min. The NaclO(4), a chaotropic agent, was added to improve peak shape and to minimize conformational effects. Retention coefficients for the amino acids were computed by using a Hewlett-Packard 9815A calculator programmed to change the retention coefficients for all amino acids sequentially to obtain a maximum correlation between actual and predicted retention times. Correlations of 0.999 at pH 7.4 and 0.997 at pH 2.1 were obtained for 25 peptides including glucagon, oxytocin, [Met]enkephalin, neurotensin, and somatostatin. This high degree of correlation suggests that, for peptides containing up to 20 residues, retention is primarily due to partition processes that involve all the residues. Although steric or conformational factors do have some effect on retention, the data suggest that under the above chromatographic conditions the retention of peptides containing up to 20 residues can be predicted solely on the basis of their amino acid composition. This possibility was tested by using data taken from the literature.