EFFECT OF PHOSPHORYLATION ON THE RETENTION BEHAVIOR OF PEPTIDES IN ION PAIRING REVERSED-PHASE HPLC BASED ON A PREDICTION MODEL

Abstract

A prediction model of the peptide retention time was developed on the basis of 358 non-phosphorylated peptides, which had various amino acid residues in the C-terminal from the casein pancreatic hydrolysates. This model was applied to predict the retention times (RT) of another 43 peptides on C4 reversed phase columns with trifluoroacetic acid (TFA) as the ion-pairing reagent, with a relatively high R² value (0.969). Furthermore, the experimental RTs of 32 phosphopeptides were compared with the predictive RTs of their non-phosphorylated cognates. Mono- and poly-phosphopeptides seemed to elute after or before the non-phosphorylated predictive cognates, respectively. The positive charges of peptides were partly masked by the ion-pairing reagent TFA. Single and multiple phosphorylation might generally lead to an increase or reduction in the overall hydrophobicity of peptides, respectively.

Keywords:

• mass spectrometry
• phosphopeptide
• phosphorylation
• prediction retention time

ACKNOWLEDGMENTS

The authors thank the financial supports from the Natural Science Foundation of China (No. 20806057) and the Ministry of Education (No. 200800561004). This research is also supported by the National High Technology Research and Development Program of China (863 Program) (No. 2008AA10Z318), and the Program for Changjiang Scholars and Innovative Research Team in University of China (IRT0641).

Notes

^a According to equation 5.

^b According to equation 6.

^a S∗corresponds to phosphoserine.

^b The number of phosphorylated residues.

^c The differences in retention time (experimental RT (phosphopeptides) − predictive RT (unphosphorylated cognates)).

^d The experimental RT of phosphopeptides.

^e The charge state of phosphopeptides in the electrospray-ionization process.

^f The number of basic residues (Lys, Arg, His).