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Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis
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A solid phase extraction-based platform for rapid phosphoproteomic analysis.

Noah Dephoure1, Steven P Gygi

  • 1Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. noah_dephoure@hms.harvard.edu

Methods (San Diego, Calif.)
|March 29, 2011
PubMed
Summary
This summary is machine-generated.

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This study presents a simplified phosphopeptide enrichment method using strong cation exchange (SCX) and immobilized metal ion affinity chromatography (IMAC). This approach accelerates phosphoproteomic analysis, enabling quantification of thousands of phosphorylation sites efficiently.

Area of Science:

  • Biochemistry
  • Proteomics
  • Cellular Biology

Background:

  • Protein phosphorylation is a critical post-translational modification regulating cellular processes.
  • Traditional methods for studying phosphorylation are time-consuming and complex.
  • Advancements in mass spectrometry have increased phosphoproteomic data but face accessibility challenges.

Purpose of the Study:

  • To develop a streamlined and cost-effective phosphopeptide enrichment strategy.
  • To reduce the technical expertise and instrument time required for phosphoproteomics.
  • To enable high-throughput quantitative analysis of phosphorylation sites.

Main Methods:

  • A modified two-step enrichment protocol involving lysC digestion.
  • Utilizing self-packed strong cation exchange (SCX) solid-phase extraction (SPE) columns with step elution.

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Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae
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Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

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Last Updated: Jun 3, 2026

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  • Integrating SCX with immobilized metal ion affinity chromatography (IMAC) followed by LC-MS/MS analysis.
  • Employing stable isotope labeling for quantitative analysis.

    • Main Results:

    • The SCX procedure is user-friendly, requires no HPLC system, and allows parallel sample processing.
    • Demonstrated efficient enrichment and quantification of over 8000 unique phosphorylation sites in yeast.
    • Achieved comprehensive phosphoproteomic analysis within 12 hours of mass spectrometry instrument time.
    • Evaluated the influence of enzyme selection and instrument platform on results.

    Conclusions:

    • The described SCX-SPE/IMAC method offers a significant time and labor saving for phosphopeptide enrichment.
    • This technique lowers the barrier to entry for phosphoproteomic studies, benefiting broader biological research.
    • The method facilitates high-throughput, quantitative phosphoproteomics, aiding disease research including cancer.