Protein Structural Characterization Using Electron Transfer Dissociation and Hydrogen Exchange-Mass Spectrometry

Intermediate states are often populated during the folding and unfolding reactions of a protein, and their detection is very challenging as they form transiently. Structural characterization of these short-lived intermediate species is difficult as it requires high-resolution methodologies. Hydrogen exchange-mass spectrometry (HX-MS) can identify and yield direct structural information on folding and unfolding intermediates, as well as information about the cooperativity of the folding or unfolding processes. The mass distributions of intact protein molecules are obtained first to determine their exchange pattern. Then, segment-specific structural information is obtained by analyzing the fragments of the protein. Enzymatic digestion is widely used with HX to determine the sequence-specific structural changes that occur to the protein during folding or unfolding. However, if a protein is an inhibitor of the protease, then alternative methodologies are required. Using electron transfer dissociation (ETD), it is possible to fragment the protein inside a mass spectrometer, and segment-specific structural changes occurring during the folding and unfolding process can be determined. In the case of HX-ETD-MS, protein molecules are first allowed to undergo HX, followed by their fragmentation. Deuterium retention in each fragment is measured. Very little, if any, scrambling of deuterium across fragments occurs during ETD-enabled fragmentation; hence, there is little scope for misinterpretation of the HX data. Key features • Analysis of intact protein data allows the identification of the intermediate states even in native and native-like conditions. • Precursor mass is determined from the intact protein analysis. • Fragments undergoing cooperative and non-cooperative transitions during protein unfolding can be distinguished from each other using HX-ETD-MS. • Analysis of the protein fragments that are obtained using ETD also enables the determination of the sequence of structural changes occurring in the protein. • HX-ETD-MS can provide structural insights into transiently formed intermediate states.