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Related Experiment Videos

Phosphoproteomics with Activated Ion Electron Transfer Dissociation.

Nicholas M Riley, Alexander S Hebert, Gerhard Dürnberger1,2,3

  • 1Institute of Molecular Pathology (IMP) , Campus-Vienna-Biocenter 1, A-1030 Vienna, Austria.

Analytical Chemistry
|April 7, 2017
PubMed
Summary

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Activated ion electron transfer dissociation (AI-ETD) significantly enhances phosphosite localization in phosphoproteomics. This method triples identifications compared to standard electron transfer dissociation (ETD) and improves accuracy for both peptides and intact proteins.

Area of Science:

  • Mass spectrometry
  • Proteomics
  • Biochemistry

Background:

  • Accurate phosphosite localization is essential for interpreting phosphoproteomic data.
  • Previous methods like electron transfer dissociation (ETD) have limitations in peptide fragmentation and identification.
  • A novel implementation of activated ion electron transfer dissociation (AI-ETD) was developed on a hybrid mass spectrometry system.

Purpose of the Study:

  • To evaluate the performance of AI-ETD for identifying and localizing phosphorylation sites.
  • To compare AI-ETD with existing methods like ETD, ETcaD, and EThcD.
  • To assess the impact of AI-ETD on intact phosphoprotein analysis.

Main Methods:

  • Utilized a quadrupole-Orbitrap-linear ion trap hybrid MS system (Orbitrap Fusion Lumos) with AI-ETD.

Related Experiment Videos

  • Analyzed phosphopeptides enriched from mouse brain lysates.
  • Investigated phosphosite localization in the intact phosphoprotein α-casein.
  • Modified the phosphoRS algorithm to account for AI-ETD-induced neutral losses.

    • Main Results:

    • AI-ETD identified 24,503 localized phosphopeptide spectral matches, more than tripling standard ETD results.
    • AI-ETD demonstrated superior performance over ETcaD and EThcD.
    • Improved fragmentation quality and MS/MS success rates were observed across all precursor charge states.
    • Modified phosphoRS showed improved localization accuracy for AI-ETD spectra.
    • AI-ETD successfully localized all eight known phosphosites in α-casein, providing the greatest sequence coverage.

    Conclusions:

    • AI-ETD significantly advances phosphoproteomics by enhancing phosphosite identification and localization.
    • The method offers substantial improvements for both bottom-up and top-down phosphoproteomic workflows.
    • AI-ETD is a powerful tool for detailed phosphoproteome analysis.