Oslund, R.C. et al. J. Am. Chem. Soc. 136, 12899–12911 (2014).

Mass spectrometry–based proteomic methods for detecting phosphorylation of serine, threonine and tyrosine are becoming well established, but this important regulatory post-translational modification (PTM) is also known to occur on other amino acid residues such as histidine. Oslund et al. describe a method for global proteomic profiling of phosphohistidine proteins, which they applied to study differences in Escherichia coli cells grown on glycerol versus on mannitol. First, to enrich for peptides containing this PTM, they utilized a pan-phosphohistidine antibody. Next, they found that collision-induced dissociation fragmentation of phosphohistidine-containing peptides in the mass spectrometer produced characteristic neutral-loss species. This observation allowed them to develop a software tool to identify phosphohistidine-containing peptides in mass spectrometry data by their characteristic fragmentation pattern.