Slow Histidine H/D Exchange Protocol for Thermodynamic Analysis of Protein Folding and Stability using Mass Spectrometry

摘要

Described here is a mass spectrometry based protocol to study the thermodynamic stability of proteins and protein-ligand complexes using the slow H/D exchange reaction of the imidazole C2 proton in histidine side chains. The protocol, which involves evaluating the denaturant dependence of this slow H/D exchange reaction in proteins, allows the global and/or subglobal unfolding/refolding properties of proteins and protein-ligand complexes to be probed. The protocol is developed using several model protein systems including: ribonuclease (Rnase) A, myoglobin, bovine carbonic anhydrase (BCA) II, hemoglobin, and the hemoglobin-haptoglobin protein complex. The compatibility of the protocol with conventional mass spectrometry-based proteomic sample preparation and analysis methods is also evaluated in an experiment in which the protocol is applied to proteins in a yeast cell lysate and used to detect the binding of Zn to superoxide dismutase in the yeast cell lysate sample. The yeast cell sample analyses also helped define the scope of the technique, which requires the presence of globally protected histidine residues in a protein’s three-dimensional structure for successful application.