Abstract
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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Acknowledgments
This work was supported by the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research No. 17H03605 and No. 17H05667, Japan Science and Technology Agency A-STEP program No. AS2915165U (Y. I.) and by grant from the Natural Sciences and Engineering Research Council of Canada (RGPIN-2016-05963; O. V. K.).
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Ogata, K., Krokhin, O.V. & Ishihama, Y. Retention Order Reversal of Phosphorylated and Unphosphorylated Peptides in Reversed-Phase LC/MS. ANAL. SCI. 34, 1037–1041 (2018). https://doi.org/10.2116/analsci.18SCP11
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DOI: https://doi.org/10.2116/analsci.18SCP11