Supplementary Figure 8 : Validation of pHis with synthetic peptides

From: Widespread bacterial protein histidine phosphorylation revealed by mass spectrometry-based proteomics

Supplementary Figure 8

Validation of histidine phosphorylation by comparison of the tandem MS spectrum derived from the endogenous peptide to spectra derived from synthetic peptides: example of the phosphosite H89 of the autonomous glycyl radical cofactor protein (grcA). (A) MS2 spectrum acquired for the endogenous E. coli phosphopeptide. (B) MS2 spectrum acquired for the synthetic histidine phosphorylated peptide RETLEDAVKpHPEK. (C) MS2 spectrum acquired for the synthetic threonine phosphorylated REpTLEDAVKHPEK isoform. Comparison of the different spectra clearly confirms the correct assignment of histidine phosphorylation of the endogenous peptide (A). The 3 different MS2 profiles were each observed in 3 different spectra.