Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry

Authors

  • Uma K. Aryal,

    Corresponding author
    1. National Research Council, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9
    Current affiliation:
    1. Pacific Northwest National Laboratory, P.O. Box 999, 902 Battelle Boulevard, Richland, WA 99352, USA.
    • Pacific Northwest National Laboratory, P.O. Box 999, 902 Battelle Boulevard, Richland, WA 99352, USA.
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  • Andrew R. S. Ross

    1. National Research Council, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9
    Current affiliation:
    1. Fisheries and Oceans Canada, Institute of Ocean Sciences, P.O. Box 6000, 9860 West Saanich Road, Sidney, British Columbia, Canada V8L 4B2.
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Abstract

Titanium dioxide metal oxide affinity chromatography (TiO2-MOAC) is widely regarded as being more selective than immobilized metal-ion affinity chromatography (IMAC) for phosphopeptide enrichment. However, the widespread application of TiO2-MOAC to biological samples is hampered by conflicting reports as to which experimental conditions are optimal. We have evaluated the performance of TiO2-MOAC under a wide range of loading and elution conditions. Loading and stringent washing of peptides with strongly acidic solutions ensured highly selective enrichment for phosphopeptides, with minimal carryover of non-phosphorylated peptides. Contrary to previous reports, the addition of glycolic acid to the loading solution was found to reduce specificity towards phosphopeptides. Base elution in ammonium hydroxide or ammonium phosphate provided optimal specificity and recovery of phosphorylated peptides. In contrast, elution with phosphoric acid gave incomplete recovery of phosphopeptides, whereas inclusion of 2,5-dihydroxybenzoic acid in the eluant introduced a bias against the recovery of multiply phosphorylated peptides. TiO2-MOAC was also found to be intolerant of many reagents commonly used as phosphatase inhibitors during protein purification. However, TiO2-MOAC showed higher specificity than immobilized gallium (Ga3+), immobilized iron (Fe3+), or zirconium dioxide (ZrO2) affinity chromatography for phosphopeptide enrichment. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was more effective in detecting larger, multiply phosphorylated peptides than liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), which was more efficient for smaller, singly phosphorylated peptides. Copyright © 2009 Crown in the right of Canada. Published by John Wiley & Sons, Ltd.

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