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From Phosphoproteome to Modeling of Plant Signaling Pathways

  • Maksim Zakhartsev
  • Heidi Pertl-Obermeyer
  • Waltraud X. Schulze
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1394)

Abstract

Quantitative proteomic experiments in recent years became almost routine in many aspects of biology. Particularly the quantification of peptides and corresponding phosphorylated counterparts from a single experiment is highly important for understanding of dynamics of signaling pathways. We developed an analytical method to quantify phosphopeptides (pP) in relation to the quantity of the corresponding non-phosphorylated parent peptides (P). We used mixed-mode solid-phase extraction to purify total peptides from tryptic digest and separated them from most of the phosphorous-containing compounds (e.g., phospholipids, nucleotides) which enhances pP enrichment on TiO2 beads. Phosphoproteomic data derived with this designed method allows quantifying pP/P stoichiometry, and qualifying experimental data for mathematical modeling.

Key words

Phosphopeptide enrichment Mixed-mode solid-phase extraction Metal oxide affinity chromatography Mathematical modeling 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Maksim Zakhartsev
    • 1
  • Heidi Pertl-Obermeyer
    • 1
  • Waltraud X. Schulze
    • 1
  1. 1.Plant Systems Biology, Plant PhysiologyUniversity of HohenheimStuttgartGermany

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