Hydrogen Exchange

Abstract

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 ( 3 H). Subsequently, the advent of onedimensional 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 highresolution 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

Interlaboratory Studies Using the NISTmAb to Advance Biopharmaceutical Structural Analytics

“…Main effects analyses revealed non-ideal mean responses for variables, , Lab# , and %D 2 O. Since amides undergo ≈3 × increases in exchange rates for each increment of 10 o C, ( Jensen and Rand, 2016 ), should increase smoothly with increasing . Contrarily, a jagged response pattern is observed for laboratories reporting = 22 o C, 24 and 25 o C, suggesting that the reported exchange bath temperatures may differ from the true temperatures.…”

Deuteros: software for rapid analysis and visualization of data from differential hydrogen deuterium exchange-mass spectrometry

“…Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a structural technique which has garnered attention for its ability to assess protein-protein and protein-ligand interactions, protein folding, and the associated dynamics of these processes (Jensen and Rand, 2016; Konermann et al , 2011; Masson et al , 2017; Mistarz et al , 2016). The basis of HDX-MS relies on the exchange of labile amide hydrogens of the protein backbones, for bulk deuterium within solution.…”

Rapid Hydrogen–Deuterium Exchange in Liquid Droplets

“…HDX can be acid, base, and/or water catalyzed. 6,32 Therefore, we hypothesize that the observed changes of rates compared to bulk measurements arise from local pH shifts in the droplets on the surface, 33,34 caused by a large number of positive charges deposited in the droplets through the ESI mechanism or through concentration redistribution of the positively charged analytes to the surface. 22,35…”