Hydrogen Exchange: A Sensitive Analytical Window into Protein Conformation and Dynamics

Hydrogen exchange (HX) monitored by mass spectrometry (MS) is a powerful analytical method for investigation of protein conformation and dynamics. HX‐MS monitors isotopic exchange of hydrogens in protein backbone amides and thus serves as a sensitive method for probing protein conformation and dynamics along the entire protein backbone (except for proline) (Figure 1.1). Historically, the monitoring of isotopic exchange in proteins has posed a technical challenge. Initial methodologies employed include measuring HX using the ultracentrifugation procedure of Kaj Ulrik Linderstrøm‐Lang [3] and later on infrared [4] or UV spectroscopy [5]. These protocols were labor‐intensive and only capable of measuring the summed (global) HX of labile sites in the protein. In the 1960s, Englander et al. [6] developed a method for monitoring isotopic exchange by liquid scintillation using the radioactive isotope, tritium (H). Subsequently, the advent of one‐ dimensional nuclear magnetic resonance (NMR) spectroscopy enabled the measurement of HX at spectrally resolved amide linkages. The impact of the latter two approaches was, however, limited. HX studies of proteins underwent a significant resurgence following the development of high‐ resolution two‐dimensional NMR methods. NMR is capable of resolving the majority of amide hydrogen signals of smaller proteins, thus increasing the number of amides through which to probe local conformational properties [7]. The combination of HX and multidimensional NMR spectroscopy