Widespread bacterial protein histidine phosphorylation revealed by mass spectrometry-based proteomics

  title={Widespread bacterial protein histidine phosphorylation revealed by mass spectrometry-based proteomics},
  author={Clement Potel and Miao-Hsia Lin and Albert J. R. Heck and Simone Lemeer},
  journal={Nature Methods},
For decades, major difficulties in analyzing histidine phosphorylation have limited the study of phosphohistidine signaling. Here we report a method revealing widespread and abundant protein histidine phosphorylation in Escherichia coli. We generated an extensive E. coli phosphoproteome data set, in which a remarkably high percentage (∼10%) of phosphorylation sites are phosphohistidine sites. This resource should help enable a better understanding of the biological function of histidine… 

Gaining Confidence in the Elusive Histidine Phosphoproteome

A mass-spectrometry-based approach that outperforms classical HCD fragmentation without compromising sensitivity is presented, using the phosphohistidine immonium ion as a diagnostic tool as well as ETD-based fragmentation techniques to achieve unambiguous identification and localization of histidine-phosphorylation sites.

Profiling of Histidine Phosphoproteome in Danio rerio by TiO2 Enrichment

This report provides the first phosphohistidine dataset obtained from zebrafish, and presents a global phosphorylation analysis of Danio rerio (zebrafish) larvae using a TiO2 affinity technique.

Widespread arginine phosphorylation in human cells - a novel protein PTM revealed by mass spectrometry

An experimental phosphoproteomic workflow is reported, which for the first time allowed to reveal the widespread occurrence of pArg in human cells by mass spectrometry and hint a possible cellular function of arginine phosphorylation by regulating the favorability of propeptide convertase substrate.

Widespread arginine phosphorylation in human cells—a novel protein PTM revealed by mass spectrometry

A phosphoproteomic workflow is reported, which allows for the first time revealing the widespread occurrence of pArg in human cells by mass spectrometry and suggests a possible cellular function of arginine phosphorylation which may regulate the favorability of propeptide convertase substrate.

Chemoproteomic Profiling of Phosphoaspartate Modifications in Prokaryotes.

A nucleophilic, desthiobiotin-containing hydroxylamine (DBHA) chemical probe that covalently labels modified aspartic acid residues in native proteomes is reported, allowing global quantification of changes in endogenous protein modification status.

Clostridioides difficile Phosphoproteomics Shows an Expansion of Phosphorylated Proteins in Stationary Growth Phase

Analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile shows that phosphorylated on serine residues is most common, followed by threonine and tyrosine phosphorylations, which forms the basis for a further investigation into the contributions of individual kinases to the overall phosphoproteome of C. difficiles.

Widespread Arginine Phosphorylation in Staphylococcus aureus

Pyrophosphoproteomics: extensive protein pyrophosphorylation revealed in human cell lines

Protein pyrophosphorylation emerges as an archetype of non-canonical phosphorylation, and should be considered in future phosphoproteomic analyses.

Characterizing Protein Kinase Substrate Specificity Using the Proteomic Peptide Library (ProPeL) Approach

The ProPeL approach allows for detailed characterization of wildtype kinase specificity motifs, identification of specificity drift due to kinase mutations, and evaluation of kinase residue structure‐function relationships.



Mass spectrometric analysis of protein histidine phosphorylation

Protein histidine phosphorylation is now recognized as an important form of post-translational modification and phosphohistidine-containing phosphopeptides derived from proteolytic digestion ofosphohistone H4 are detectable by ESI-MS and reverse-phase HPLC separation is demonstrated.

Impact of phosphoproteomics on studies of bacterial physiology.

Bacterial phosphoproteomics provided interesting insights into the evolutionary aspects of protein phosphorylation and inspired a number of physiological studies in which the identity of the phosphorylated site facilitated the elucidation of molecular mechanisms of signaling and regulation.

Systematic profiling of the bacterial phosphoproteome reveals bacterium-specific features of phosphorylation

Differences in phosphorylation preferences between bacteria and eukaryotes revealed by this study could be useful in identifying bacterial-specific targets for future therapies.

A Pan-specific Antibody for Direct Detection of Protein Histidine Phosphorylation

The development of the first pan-pHis antibody using a stable phosphohistidine (pHis) mimetic as the hapten is reported, which found that pHis levels on PpsA are sensitive to nitrogen availability in vivo and that α-ketoglutarate inhibits phosphotransfer from phosphorylated phosphoenolpyruvate synthase (PpsA) to pyruvates in vivo.

Synthesis and characterization of histidine‐phosphorylated peptides

Methods for the synthesis and analysis of phosphohistidine‐containing peptides are described, a prerequisite for the investigation of the role of this posttranslational modification in cellular processes.

A Phosphohistidine Proteomics Strategy Based on Elucidation of a Unique Gas-Phase Phosphopeptide Fragmentation Mechanism

It is shown that collision-induced dissociation of pHis peptides produces prominent characteristic neutral losses of 98, 80, and 116 Da, and a software tool that screens LC–MS/MS spectra for potential matches to pHis-containing peptides based on their neutral loss pattern is developed.

Chasing phosphohistidine, an elusive sibling in the phosphoamino acid family.

The challenges associated with studying the chemical biology of pHis are discussed and recent progress is reviewed that offers some hope that long-awaited biochemical reagents for studying this elusive posttranslational modification (PTM) might soon be available.

The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry

The SCX/IMAC enrichment protocol is described, which has successfully employed in the exploration of phosphoproteomes from several systems including mouse liver and Drosophila embryos characterizing over 5,500 and 13,000 phosphorylation events, respectively.