Analysis of 1- and 3-Phosphohistidine (pHis) Protein Modification Using Model Enzymes Expressed in Bacteria

Despite the discovery of protein histidine (His) phosphorylation nearly six decades ago, difficulties in measuring and quantifying this unstable post-translational modification (PTM) have limited its mechanistic analysis in prokaryotic and eukaryotic signaling. Here, we describe reliable procedures for affinity purification, cofactor-binding analysis and antibody-based detection of phosphohistidine (pHis), on the putative human His kinases NME1 (NDPK-A) and NME2 (NDPK-B) and the glycolytic phosphoglycerate mutase PGAM1. By exploiting isomer-specific monoclonal N1-pHis and N3-pHis antibodies, we describe robust protocols for immunological detection and isomer discrimination of site-specific pHis, including N3-pHis on His 11 of PGAM1.