Histidine phosphorylation is known to play major biological roles in prokaryotes, notably in bacterial cell metabolism or in primitive signal transduction pathways. In recent years compelling evidences that histidine phosphorylation also plays important biological roles in eukaryotes gradually accumulated. Nevertheless, the lack of adequate analytical methods to study the unstable histidine phosphorylation constitutes to this day the biggest hurdle to the large-scale study of histidine phosphorylation and consequentlyto the unraveling of the full extent of its biological roles.
Here we present - to this day - the sole method capable of enriching histidine phosphorylation at the peptide level and providing site-specific localization and quantification. We demonstrate that in the right conditions, IMAC can be used to enrich histidine phosphorylation at the peptide level. In addition, we report the use of the diagnostic phosphohistidine immonium ion as a tool to unambiguously localize histidine phosphorylation. Along the way we identified nucleic acid containing biomolecules as the main contaminants present in phospho-enriched samples after affinity chromatography. We tackled this problem by developing a robust and efficient sample preparation protocol, incorporating nucleic acid enzymatic digestion and protein precipitation. This optimized protocol enabled a 10-fold increase of the number of identified phosphosites in both gram negative as well as gram negative bacteria. The coverage allowed for a comprehensive analysis of the changes in phosphoproteome in response to antibiotic treatment and in antibiotic resistance. All together these recent methodological advances presented here have led to a better insight into bacterial signaling and show that phosphorylation is far more abundant than anticipated in bacteria. In addition, our technical advances could answer a long-standing question: is histidine phosphorylation also playing a major role in higher organisms?