Integrated Strategy for Unbiased Profiling of the Histidine Phosphoproteome

Histidine
phosphorylation (pHis), which plays a key role in signal
transduction in bacteria and lower eukaryotes, has been shown to be
involved in tumorigenesis. Due to its chemical instability, substoichiometric
properties, and lack of specific enrichment reagents, there is a lack
of approaches for specific and unbiased enrichment of pHis-proteins/peptides.
In this study, an integrated strategy was established and evaluated
as an unbiased tool for exploring the histidine phosphoproteome. First,
taking advantage of the lower charge states of pHis-peptides versus
the non-modified naked peptides at weak acid solution (∼pH
2.7), strong cation exchange (SCX) chromatography was used to differentiate
modified and non-modified naked peptides. Furthermore, selective enrichment
of the pHis-peptide was performed by applying Cu-IDA beads enrichment.
Finally, stable isotope dimethyl labeling was introduced to guarantee
high-confidence assignment of pHis-peptides. Using this integrated
strategy, 563 different pHis-peptides (H = 1) in 385 proteins were
identified from HeLa lysates. Motif analysis revealed that pHis prefers
hydrophobic amino acids and has the consensus motif-HxxK, which covered
the reports from different approaches. Thus, our method may provide
an unbiased and effective tool to reveal histidine phosphoproteome
and to study the biological process and function of histidine phosphorylation.