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Phosphopeptide Enrichment from Bacterial Samples Utilizing Titanium Oxide Affinity Chromatography

  • Boumediene Soufi
  • Christoph Täumer
  • Maja Semanjski
  • Boris Macek
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1841)

Abstract

Mass spectrometry (MS)-based proteomics detected hundreds of phosphorylation sites on serine, threonine and tyrosine in numerous bacterial proteins, firmly establishing the presence and importance of this posttranslational modification in prokaryotes. Recent biological follow up of these results revealed that vital processes in bacterial cell, such as cell division, differentiation, spore germination and persistence, are regulated by protein phosphorylation, raising the need to study this modification on a global scale under additional physiological conditions. Due to low abundance and low stoichiometric levels of protein phosphorylation, initial protocols for phosphopeptide enrichment and analysis required relatively high amounts of starting material, extensive fractionation and MS measurement time. Here we present a protocol for phosphopeptide enrichment and detection based on TiO2 chromatography and high resolution MS that enables in-depth detection and quantification of phosphorylation sites from significantly lower amounts of starting material and in a fraction of MS measurement time.

Key words

Phosphoproteome Phosphorylation TiO2 chromatography Mass spectrometry Phosphopeptide enrichment 

Notes

Acknowledgments

We thank Dr. Olaf Voolstra for critical reading of the manuscript. Our work is supported by the SFB766 of the Deutsche Forschungsgemeinschaft and PRIME-XS consortium.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Boumediene Soufi
    • 1
  • Christoph Täumer
    • 1
  • Maja Semanjski
    • 1
  • Boris Macek
    • 1
  1. 1.Proteome Center TübingenUniversity of TübingenTübingenGermany

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