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

Protein phosphorylation is one of the most ubiquitous post-translational modifications in human, and trypsin-digested phosphorylated peptides have been analyzed by reversed phase LC/MS using C18-silica columns under the acidic conditions to profile human phosphoproteomes. Here, we reported that phosphopeptides generally retain stronger 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 used or more acidic and hydrophobic ion-pairing reagents such as trifluoroacetic acid are used with C18-silica columns. These phenomena could be explained by 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.