Protein phosphorylation is the primary mean of regulation of protein biological activity. As such, communication and signaling within cells is controlled by minute changes of the phosphorylation status of proteins involved. Protein histidine phosphorylation is often referred to as being part of the hidden or missing phosphoproteome. Indeed, while in recent years evidence that histidine phosphorylation plays important biological roles in eukaryotes slowly accumulated, the lack of adequate analytical methods to study the labile histidine phosphorylation constituted the biggest hurdle to the large-scale study of histidine phosphorylation and ipso facto to the unraveling of the full extent of its biological roles. When considering the known crucial biological roles of canonical Ser/Thr/Tyr phosphorylations, it becomes obvious that exploring this so-called hidden phosphoproteome could have potentially important biological implications. This is why during my PhD we focused on the development of mass spectrometry-based analytical strategies to study protein histidine phosphorylation.
We developed a unique method capable of enriching histidine phosphorylated peptides and providing site-specific localization and quantification, a prerequisite to understand the roles of histidine phosphorylation at a molecular level. To do so, we challenged a well-established dogma and demonstrated for the first time that under the right experimental conditions, immobilized metal affinity chromatography (IMAC) is suitable for the enrichment of histidine phosphorylation at the peptide level. Along the way, we identified nucleic acid-containing biomolecules as the main contaminants 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. As a result, we were able to improve the number of STY phosphorylation sites identified by more than 50% in human cell lines, and by more than 10 times in bacteria, whose phosphoproteomes are notoriously difficult to analyze.
After further optimizations allowing the preservation of histidine phosphorylation, we applied this workflow to study phosphorylation in E. coli. This resulted in the largest reported bacterial phosphoproteome to date, allowing us to see well beyond the classically observed phosphorylation events on high abundant metabolic enzymes. Remarkably, around 10% of the identified phosphosites were on histidine residues. We showed that our method is sensitive and reproducible in term of both identification and quantification of histidine phosphosites.
Commonly used collision induced dissociation methods are often not suitable to obtain unambiguous phosphosites localization. We thus investigated new approaches to confidently localize histidine phosphosites. We report that the phosphohistidine immonium ion constitutes a signature of the presence of a histidine phosphorylation within a peptide. We used this unique feature to develop a new MS-based strategy combining both the presence of the immonium ion and electron transfer dissociation-based fragmentation to provide precise identification and localization of histidine phosphosites.
In conclusion, this work paves the way for the study of the elusive histidine phosphorylation and more generally of long-neglected phosphorylation dynamics in microorganisms.