Integrated Strategy for High-Confident Global Profiling of the Histidine Phosphoproteome

Abstract

Background

Histidine phosphorylation (pHis) plays a key role in signal transduction in prokaryotes and regulates tumour initiation and progression in mammals. However, the pHis substrates and their functions are rarely known due to the lack of effective analytical strategies.

Results

Herein, we provide a strategy for unbiased enrichment and assignment of the pHis peptides. First, the entire procedure was designed under alkaline conditions to maintain the stability of the N-P bond of pHis and high-pH reverse-phase chromatography was used to efficiently separate the pHis peptides. Second, exploiting the coelution benefits of diethyl labelling, the ratios of light- and heavy-labelled peptides were accurately quantified, and the sites of phosphorylated histidine were assigned. Finally, Cu-IDA bead enrichment and data-independent acquisition mass spectrometry analysis were used to improve the coverage of the histidine phosphoproteome. With this novel strategy, 768 and 1,125 potential pHis peptides were identified from lysates of E. coli and HeLa cells, respectively. And these values represent the highest coverage of the histidine phosphoproteome for both cell types.

Significance

These data strongly support the presumption that pHis modifications are widely present in bacteria. The study provides an efficient strategy and can lead to a better understanding of pHis-modified substrates and their biological functions.