Distinguishing Histidine Tautomers in Proteins Using Covalent Labeling-Mass Spectrometry

In this work, we use diethylpyrocarbonate (DEPC)-based covalent labeling together with LC-MS/MS analysis to distinguish the two sidechain tautomers of histidine residues in peptides and proteins. From labeling experiments on model peptides, we demonstrate that DEPC reacts equally with both tautomeric forms to produce chemically different products with distinct dissociation patterns and LC retention times, allowing the ratios of the two tautomers to be determined in peptides and proteins. Upon measuring the tautomer ratios of several histidine residues in myoglobin, we find good agreement with previous 2D NMR data on this protein. Because our DEPC labeling/MS approach is simpler, faster, and more precise than 2D NMR, our method will be a valuable way to determine how protein structure enforces histidine sidechain tautomerization. Because the tautomeric state of histidine residues is often important for protein structure and function, the ability of DEPC labeling/MS to distinguish histidine tautomers should equip researchers with a tool to understand the histidine residue structure and function more deeply in proteins.

Automated summary

By using DEPC-based covalent labeling with LC-MS/MS analysis, researchers can distinguish histidine tautomers in peptides and proteins and determine their ratios. This method is simpler, faster, and more precise than 2D NMR, providing a valuable tool for understanding the structure and function of histidine residues in proteins. The ability of DEPC labeling/MS to differentiate histidine tautomers equips researchers with a deeper insight into the structure and function of histidine residues in proteins.