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

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  • OGATA Kosuke
    Graduate School of Pharmaceutical Sciences, Kyoto University
  • KROKHIN Oleg V.
    Manitoba Centre for Proteomics and Systems Biology and Department of Internal Medicine, University of Manitoba
  • ISHIHAMA Yasushi
    Graduate School of Pharmaceutical Sciences, Kyoto University

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Abstract

<p>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.</p>

Journal

  • Analytical Sciences

    Analytical Sciences 34 (9), 1037-1041, 2018-09-10

    The Japan Society for Analytical Chemistry

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