Utility of Retention Prediction Model for Investigation of Peptide Separation Selectivity in Reversed-Phase Liquid Chromatography: Impact of Concentration of Trifluoroacetic Acid, Column Temperature, Gradient Slope and Type of Stationary Phase

摘要

Peptide separation selectivity in reversed-phase liquidrnchromatography was investigated using a training set ofrn165 peptides with a total of 1698 amino acid residues.rnGradient separation was performed at selected chromatographicrnconditions, varying column temperature, gradientrnslope, ion-pairing reagent concentration, and type ofrnstationary phase. The retention times for each set ofrnexperiments were utilized to calculate the amino acidrnretention coefficients using a published prediction modelrn(Rapid Commun. Mass Spectrom. 2007, 21, 2813-rn2821). The calculated retention coefficients reflect therncontribution of each type of amino acid residue to peptidernretention at a given chromatographic condition. For example,rnthe concentration of ion-pairing reagent (trifluoroaceticrnacid) had the strongest impact on the retention ofrnpeptides containing an increasing number of basic (charged)rnamino acids, such as arginine, lysine, and histidine, whilernthe retention coefficients of other amino acids are minimallyrnaffected. Increasing the separation temperature resulted inrna moderate decrease of amino acid retention coefficientsrnwith the exception of isoleucine, leucine, valine, and proline.rnThis finding suggests that these residues enhance peptidernretention at elevated temperature. Amino acid residuernretention coefficients were also helpful to understand thernimpact of sorbent pore size (130 vs 300 Å) on peptidernretention selectivity. In addition we investigated the selectivityrndifferences of various reversed-phase chromatographicrnsorbents. The trends suggested by calculated retentionrncoefficients were confirmed using a set of synthetic peptidesrnwith specifically designed sequences.