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.近年来，技术革新使得我们得以探究此前一直难以捉摸的蛋白质组氨酸磷酸化（protein histidine phosphorylation）。然而，对该修饰进行可靠的位点特异性定位仍颇具挑战。为此，我们提出了一种基于质谱（mass spectrometry）的分析方法，该方法在不牺牲检测灵敏度的前提下，性能优于传统的HCD碎裂技术。我们将磷酸组氨酸亚胺离子（phosphohistidine immonium ion）作为诊断工具，并结合基于ETD的碎裂技术，实现了组氨酸磷酸化位点的精准鉴定与定位。本研究工作将助力对磷酸组氨酸蛋白质组开展更可靠的探究，以揭示组氨酸磷酸化在细胞信号传导事件中的作用。