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.
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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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Soufi, B., Täumer, C., Semanjski, M., Macek, B. (2018). Phosphopeptide Enrichment from Bacterial Samples Utilizing Titanium Oxide Affinity Chromatography. In: Becher, D. (eds) Microbial Proteomics. Methods in Molecular Biology, vol 1841. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8695-8_16
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DOI: https://doi.org/10.1007/978-1-4939-8695-8_16
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