Real-time hydrogen/deuterium exchange kinetics via supercharged electrospray ionization tandem mass spectrometry

Abstract

Amide hydrogen/deuterium exchange (HDX) rate constants of bovine ubiquitin in an ammonium acetate solution containing 1% of the electrospray ionization (ESI) "supercharging" reagent m-nitrobenzyl alcohol (m-NBA) were obtained using top-down, electron transfer dissociation (ETD) tandem mass spectrometry (MS). The supercharging reagent replaces the acid and temperature "quench" step in the conventional MS approach to HDX experiments by causing rapid protein denaturation to occur in the ESI droplet. The higher charge state ions that are produced with m-NBA are more unfolded, as measured by ion mobility, and result in higher fragmentation efficiency and higher sequence coverage with ETD. Single amino acid resolution was obtained for 44 of 72 exchangeable amide sites, and summed kinetic data were obtained for regions of the protein where adjacent fragment ions were not observed, resulting in an overall spatial resolution of 1.3 residues. Comparison of these results with previous values from NMR indicates that the supercharging reagent does not cause significant structural changes to the protein in the initial ESI solution and that scrambling or back-exchange is minimal. This new method for top-down HDX-MS enables real-time kinetic data measurements under physiological conditions, similar to those obtained using NMR, with comparable spatial resolution and significantly better sensitivity.

Figure 1

Plot of the (a) ESI-MS relative charge state abundances of 10 µM ubiquitin (20 mM aqueous ammonium acetate, pH 7.0) formed with and without 1% m-NBA, and (b) TWIMS reduced arrival times as a function of charge state, which shows two distinct families of conformers.

Figure 2

Electron transfer dissociation mass spectra of the isolated (a) 7+ and (b) 10+ ions obtained from a 3.75 µM ubiquitin, 200 mM ammonium acetate, pH 6.2, solution containing 0% and 1% m-NBA, respectively. In both cases, these were the highest charge states that could be reliably isolated with sufficient S/N to perform ETD. The reduced precursors are indicated with a *. Insets are expansions of the m/z 827 – 877 regions to illustrate differences in fragmentation efficiencies.

Figure 3

Sequence coverage of ubiquitin obtained from ETD of the 7+ and 10+ charge states of 3.75 µM ubiqutin formed by electrospray from a 200 mM ammonium acetate, pH 6.2, solution containing 0% and 1% m-NBA, respectively. 25% and 86% sequence coverage was obtained for the 7+ and 10+, respectively.

Figure 4

Partial ETD mass spectra of fully deuterated 10+ ubiquitin showing the c₁₇ (left) and c₁₆ (right) fragment ions obtained at 3, 41, and 92 minutes during exchange for hydrogen. The monoisotopic peak (m) and each isotope peak in the distribution are labeled. Partial ETD spectra for the fully protonated c₁₇ and c₁₆ fragment ions obtained without HDX are inset.

Figure 5

Example hydrogen/deuterium exchange kinetics, where single residue resolution was obtained for (a) Val5 and Glu18 and (b) a three-residue sum and a two-residue sum were obtained for Leu67, His68, and Leu69, and Gly75 and Gly76, respectively. Single residue resolution results for (c) Asn25 and Thr55 illustrate decay to different average deuterium content endpoints. Exchange kinetics for (d) Asp 32 plotted with c ion data (red) and z• ion data (black), and as part of a three-residue in silico digestion (Ile30 + Gln31 + Asp32). Data were fit to single or double exponential functions where appropriate.

Figure 6

Comparison of “Relative Protection” from HDX for individual residues obtained by MS (red diamonds), NMR (black squares), and NMR (black triangles) across the protein sequence. “Relative Protection” is calculated as the (negative) logarithm of the exchange rate constants normalized within a data set.