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Related Experiment Video

Updated: Feb 5, 2026

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis
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High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform.

Sean J Humphrey1,2, Ozge Karayel3, David E James4,5,6

  • 1School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales, Australia. sean.humphrey@sydney.edu.au.

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|September 8, 2018
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Summary

The EasyPhos workflow streamlines phosphoproteomics, enabling deep analysis of hundreds of samples with minimal protein input. This method enhances reproducibility and reduces preparation time for global cell signaling studies.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Signaling

Background:

  • Mass spectrometry has revolutionized cell signaling research through phosphoproteomics.
  • Advancements enable sophisticated studies, yet practical challenges persist, limiting throughput and sample efficiency.

Purpose of the Study:

  • To present an updated EasyPhos workflow for high-performance phosphoproteomics.
  • To enhance sample efficiency and reduce preparation time, especially for limited sample amounts.

Main Methods:

  • Eliminated protein precipitation steps.
  • Performed entire protocol, including digestion, in a single 96-well plate.
  • Utilized a streamlined workflow for mass spectrometry analysis.

Main Results:

  • Achieved high reproducibility with a small sample size requirement (≤200 μg protein).
  • Reduced sample preparation time to 1 day, with mass spectrometry analysis at ~1 hour per sample.
  • Quantified 20,132 distinct phosphopeptides from 200 μg of glioblastoma protein, identifying thousands of EGF-regulated phosphorylation events.

Conclusions:

  • The updated EasyPhos workflow significantly improves phosphoproteomics efficiency and reproducibility.
  • Enables large-scale analysis of hundreds of phosphoproteomes, even with limited starting material.
  • Facilitates deep insights into cell signaling pathways, such as EGF-regulated events in glioblastoma.