Gaining Confidence in the Elusive Histidine Phosphoproteome

Recent technological
advances have made it possible to investigate
the hitherto rather elusive protein histidine phosphorylation. However,
confident site-specific localization of protein histidine phosphorylation
remains challenging. Here, we address this problem, presenting a mass-spectrometry-based
approach that outperforms classical HCD fragmentation without compromising
sensitivity. We use 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.
The work presented here will allow more confident investigation of
the phosphohistidine proteome to reveal the roles of histidine phosphorylation
in cellular signaling events.