Requirements for prediction of peptide retention time in reversed-phase high-performance liquid chromatography: hydrophilicity/hydrophobicity of side-chains at the N- and C-termini of peptides are dramatically affected by the end-groups and location

Abstract

The value of reversed-phase high-performance liquid chromatography (RP-HPLC) and the field of proteomics would be greatly enhanced by accurate prediction of retention times of peptides of known composition. The present study investigates the hydrophilicity/hydrophobicity of amino acid side-chains at the N- and C-termini of peptides while varying the functional end-groups at the termini. We substituted all 20 naturally occurring amino acids at the N- and C-termini of a model peptide sequence, where the functional end-groups were N(alpha)-acetyl-X- and N(alpha)-amino-X- at the N-terminus and -X-C(alpha)-carboxyl and -X-C(alpha)-amide at the C-terminus. Amino acid coefficients were subsequently derived from the RP-HPLC retention behaviour of these peptides and compared to each other as well as to coefficients determined in the centre of the peptide chain (internal coefficients). Coefficients generated from residues substituted at the C-terminus differed most (between the -X-C(alpha)-carboxyl and -X-C(alpha)-amide peptide series) for hydrophobic side-chains. A similar result was seen for the N(alpha)-acetyl-X- and N(alpha)-amino-X- peptide series, where the largest differences in coefficient values were observed for hydrophobic side-chains. Coefficients derived from substitutions at the C-terminus for hydrophobic amino acids were dramatically different compared to internal coefficients for hydrophobic side-chains, ranging from 17.1 min for Trp to 4.8 min for Cys. In contrast, coefficients derived from substitutions at the N-terminus showed relatively small differences from the internal coefficients. Subsequent prediction of peptide retention time, within an error of just 0.4 min, was achieved by a predictive algorithm using a combination of internal coefficients and coefficients for the C-terminal residues. For prediction of peptide retention time, the sum of the coefficients must include internal and terminal coefficients.

Figure 1

Panel A: Correlation plot of the hydrophilicity/hydrophobicity coefficients at the peptide C-terminus when the end group is C^α-amide versus C^α-carboxyl. The side-chain coefficients were determined from reversed-phase retention behaviour of peptides at 25°C (data taken from Peptide 1 and Peptide 2, Table 3). The peptide sequences are shown in Table 1. The hydrophobic amino acid substitutions are denoted by the single letter code. Y = 0.834 x + 0.522, correlation coefficient R = 0.993. Panel B: The differences in coefficients between Peptide 1 and Peptide 2 are plotted as a bar graph (data taken from Table 3).

Figure 2

Panel A: Correlation plot of the hydrophilicity/hydrophobicity coefficients at the peptide N-terminus when the end group is N^α-amino versus N^α-acetyl. The coefficients were determined from reversed-phase retention behaviour of peptides at 25°C (data taken from Peptide 3 and Peptide 4, Table 3). The peptide sequences are shown in Table 1. The hydrophobic amino acid substitutions are denoted by the single letter code. Y = 0.739 x + 0.114, correlation coefficient R = 0.982. Panel B: The difference in coefficients between Peptide 3 and Peptide 4 are plotted as a bar graph (data taken from Table 3).

Figure 3

Panel A: Correlation plot of the hydrophilicity/hydrophobicity coefficients at the peptide N-terminus with a N^α-amino group versus coefficients at the C-terminus with a C^α-carboxyl group determined from reversed-phase retention behaviour of peptides at 25°C (data taken from Peptide 1 and Peptide 4, Table 3). The peptide sequences are shown in Table 1. The hydrophobic amino acid substitutions are denoted by the single letter code. Y = 0.558 x + 0.242, correlation coefficient R = 0.967. Panel B: The differences in coefficients between Peptide 1 and Peptide 4 are plotted as a bar graph (data taken from Table 3).

Figure 4

Panel A: Correlation plot of the hydrophobicity/hydrophilicity coefficients at the C-terminus with a C^α-carboxyl group versus internal side-chain coefficients (data taken from Peptide 1 and Peptide 5, Table 3). The peptide sequences are shown in Table 1. The hydrophobic amino acid side-chains are denoted by the single letter code. Y = 0.985 x − 0.497, correlation coefficient R = 0.984. Panel B: The difference in coefficients between Peptide 1 and Peptide 5 are plotted as a bar graph (data taken from Table 3).

Figure 5

Panel A: Correlation plots of predicted retention times versus observed retention times of the data shown in Table 4 for columns 2, 4 and 6. Plot A uses the C-terminal X-carboxyl coefficients for all residues in the peptide sequence; Plot B uses the weighted C-terminal carboxyl coefficients for all residues in the peptide sequence; Plot C uses the weighted coefficients for all residues except the C-terminal residue which uses the C-terminal X-carboxyl coefficients. Panel B: Correlation plots of the difference between predicted and observed versus observed retention times. Plots A, B and C use predicted values determined as described in Panel A.

Figure 6

Panel A: Correlation plots of predicted retention times versus observed retention times for the data shown in Table 4 for columns 8 and 10. Plot A uses the experimentally derived internal coefficients for all residues in the sequence and Plot B uses the internal coefficients for all residues except the C-terminal residue which uses the C-terminal X-carboxyl coefficients. Panel B: Correlation plots of the difference between predicted and observed versus observed retention times. Plots A and B use predicted values determined as described in Panel A.