Gaining confidence in the elusive histidine phosphoproteome

Abstract

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 outperforming 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 the more confident investigation of the phosphohistidine proteome to reveal the roles of histidine phosphory-lation in cellular signaling events.