Temperature Dependence of Retention Behavior of Phosphorylated Peptides in Ion-Pair Reversed-Phase Liquid Chromatography

The success of the phosphoproteome analysis depends on the separation efficiency of phosphorylated peptides in ion-pair reversed-phase liquid chromatography coupled with tandem mass spectrometry. Here, we report that the phosphorylated peptides were less susceptible to the column temperature than the unphosphorylated peptides when trifluoroacetic acid (TFA) was used as an ion-pair reagent. This trend was partially reversed when TFA was replaced by acetic acid, and the retention order of phosphorylated and unphosphorylated peptide pairs was also partially reversed. Our results indicate that the retention behavior of phosphorylated peptides is relatively predictable in the TFA system, which has long been used in ion-pair liquid chromatographic analysis of peptides, but in the acetic acid system, which is compatible with mass spectrometry, the retention behavior is more diverse, and further data accumulation is needed to quantitatively understand the retention behavior.