Analytical Sciences

Retention Order Reversal of Phosphorylated and Unphosphorylated Peptides in Reversed-Phase LC/MS

Kosuke OGATA,* Oleg V. KROKHIN,** and Yasushi ISHIHAMA*

*Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshidashimoadachicho, Sakyo, Kyoto 606-8501, Japan
**Manitoba Centre for Proteomics and Systems Biology and Department of Internal Medicine, University of Manitoba, 799 JBRC, 715 McDermot Avenue, Winnipeg, R3E 3P4, Canada

Protein phosphorylation is one of the most ubiquitous post-translational modifications in humans, and trypsin-digested phosphorylated peptides have been analyzed by reversed phase LC/MS using C18-silica columns under acidic conditions to profile human phosphoproteomes. Here, we report that phosphopeptides generally exhibit stronger retention than their unphosphorylated counterparts when C18-silica columns are used with acetic acid or formic acid as an ion-pairing reagent, whereas the retention order is reversed when less hydrophobic stationary phases such as C4-silica columns are employed. Similarly the retention reversal is observed when more hydrophobic ion-pairing reagents such as trifluoroacetic acid are used with C18-silica columns. These phenomena could be explained by the smaller S-values of phosphopeptides in linear solvation strength theory, based on the reduced net charge caused by intramolecular interaction between phosphate and basic groups.

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