Measuring the Hydrogen/Deuterium Exchange of Proteins at High Spatial Resolution by Mass Spectrometry: Overcoming Gas-Phase Hydrogen/Deuterium Scrambling

Abstract

Proteins are dynamic molecules that exhibit conformational flexibility to function properly. Well-known examples of this are allosteric regulation of protein activity and ligand-induced conformational changes in protein receptors. Detailed knowledge of the conformational properties of proteins is therefore pertinent to both basic and applied research, including drug development, since the majority of drugs target protein receptors and a growing number of drugs introduced to the market are therapeutic peptides or proteins. X-ray crystallography provides a static picture at atomic resolution of the lowest-energy structure of the native ensemble. There is a growing need for sensitive analytical tools to explore all of the significant molecular structures in the conformational landscape of proteins. Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has recently emerged as a powerful method for characterizing protein conformational dynamics. The basis of this method is the fact that backbone amides in stable hydrogen-bonded structures (e.g., α-helices and β-sheets) are protected against exchange with the aqueous solvent. All protein structures are dynamic, however, and eventually all of the protecting hydrogen bonds will transiently break as the protein--according to thermodynamic principles--cycles through partially unfolded states that correspond to excited free energy levels. As a result, all of the backbone amides will eventually become temporarily solvent-exposed and exchange-competent over time. Consequently, a folded protein in D2O will gradually incorporate deuterium into its backbone amides, and the kinetics of the process can be readily monitored by mass spectrometry. The deuterium uptake kinetics for the intact protein (global exchange kinetics) represents the sum of the exchange kinetics for the individual backbone amides. Local exchange kinetics is typically achieved by using pepsin digestion under quench conditions (i.e., under cold acidic conditions where the amide hydrogen exchange rate is slowed by many orders of magnitude). The ability to localize the individual deuterated residues (the spatial resolution) is determined by the size (typically ∼7-15 residues) and the number of peptic peptides. These peptides provide a relatively coarse-grained picture of the protein dynamics. A fundamental understanding of the relationship between protein function/dysfunction and conformational dynamics requires in many cases higher resolution and ultimately single-residue resolution. In this Account, we summarize our efforts to achieve single-residue deuterium levels in proteins by electron-based or laser-induced gas-phase fragmentation methods. A crucial analytical requirement for this approach is that the pattern of deuterium labeling from solution is retained in the gas-phase fragment ions. It is therefore essential to control and minimize any occurrence of gas-phase randomization of the solution deuterium label (H/D scrambling) during the MS experiment. For this purpose, we have developed model ...

Sites Involved in Intra- and Interdomain Allostery Associated with the Activation of Factor VIIa Pinpointed by Hydrogen-Deuterium Exchange and Electron Transfer Dissociation Mass Spectrometry

“…The mass spectrometer was equipped with an ESI source operated at predefined settings for minimal H/D scrambling as described previously (11,12,(31)(32)(33)(34)(35). ETD was performed in the trap T-wave by reacting radical 1,4-dicyanobenzene anions with quadrupole-selected deuterium-labeled peptide ions.…”

“…Mass spectrometry (MS) has evolved to be a powerful technique to measure protein HDX and thus monitor protein dynamics in solution, due to tolerance to complex protein systems, buffer composition, and low sample concentration. More recently, the integration of electron transfer dissociation (ETD) into the HDX-MS workflow has enabled the mapping of conformational changes in proteins at a spatial resolution down to individual residues (11,12,(31)(32)(33)(34)(35). During HDX-ETD measurements, peptides generated from solution-phase proteolysis are further fragmented in the gas-phase by ETD, and through mass analysis of fragment ions deuterium contents can often be assigned to individual sites.…”

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

“…Native electrospray ionization (ESI)-MS and ion mobility spectrometry yield information on quaternary structure and subunit connectivities [2][3][4]. Hydrogen/deuterium exchange reports on secondary structure and dynamics [5][6][7][8]. Cross-linking provides distance constraints that can be essential for structure determination efforts [9][10][11][12][13].…”

Mapping Residual Structure in Intrinsically Disordered Proteins at Residue Resolution Using Millisecond Hydrogen/Deuterium Exchange and Residue Averaging

“…Still, this approach cannot match the spatial resolution achievable using nuclear magnetic resonance (NMR). For this reason, experimental [16][17][18] or computational [19][20][21] approaches that provide single residue resolution are highly desirable.…”