H/D exchange and mass spectrometry in the studies of protein conformation and dynamics: is there a need for a top-down approach?

Abstract

Hydrogen/deuterium exchange (HDX) combined with mass spectrometry (MS) detection has matured in recent years to become a powerful tool in structural biology and biophysics. Several limitations of this technique can and will be addressed by tapping into the ever expanding arsenal of methods to manipulate ions in the gas phase offered by mass spectrometry.

Figure 1

Schematic representation of HDX MS experiments: bottom-up (A) and top-down (B) HDX MS.

Figure 2

HDX MS of Fe₂Tf in the presence (blue) and the absence (red) of the cognate receptor. The exchange was carried out by diluting the protein stock solution 1:10 in exchange solution (100 mM NH₄HCO₃ in D₂O, pH adjusted to 7.4) and incubating for a certain period of time as indicated on each diagram followed by rapid quenching (lowering pH to 2.5 and temperature to near 0°C). The black trace shows unlabeled protein.

Figure 3

Localizing the influence of the receptor binding on backbone protection of Fe₂Tf using bottom-up HDX MS on the physiologically relevant time scale. The panels show isotopic distributions of representative peptic fragments derived from the protein subjected to HDX in the presence (blue) and the absence (red) of the receptor and followed by rapid quenching. Dotted lines indicate deuterium content of unlabeled and fully exchanged peptides. Colored segments within the Fe₂Tf/receptor complex show location of the peptic fragments.

Figure 4

Characterization of local dynamics in wt*-CRABP I in a conformer-specific fashion using top-down HDX MS (fully deuterated protein was exposed to ¹H₂O/CH₃CO₂N¹H₄ at pH 3.1 for 10 min; the gray trace at the bottom corresponds to HDX end-point). A: mass selection of precursor ions for subsequent CAD (from top to bottom): broad-band selection of the entire ionic population (not conformer-specific); highly protected conformers; narrow population of less protected conformers; HDX end-point. B: isotopic distributions of two representative fragment ions generated by CAD of precursor ions shown in panel A. Selection of different ion populations as precursor ions for subsequent fragmentation was achieved by varying the width of a mass selection window of a quadrupole filter (Q) in a hybrid quadrupole/time-of-flight mass spectrometer (Qq-TOF MS).

Figure 5

Top-down HDX MS of wt*-CRABP I using ECD of the entire protein ion population (fully deuterated protein was exposed to ¹H₂O/CH₃CO₂N¹H₄ at pH 3.5 for varying time periods); the black trace at the bottom of corresponds to HDX end-point). A: isotopic distributions of surviving intact protein ions. B: two representative c-ions. Minimal collision-and temperature-induced desolvation was used for acquisition of all mass spectra, except the one top (red trace).

Figure 6

Backbone protection pattern of CRABPI mutant (without N-terminal His-tag) obtained from top-down HDX MS measurements using ETD of the entire protein ion population. HDX was initiated by exposing the fully deuterated protein to ¹H₂O/CH₃CO₂N¹H₄ at pH 3.5 for 5 min followed by rapid quenching.

Figure 7

An example of raw HDX MS data used to generate the protection plot shown in Figure 6. Isotopic distributions of c₁₃ and c₃₄ fragments derived from protein subjected to 5 min HDX exchange in solution (red trace) and protein at the HDX end-point (blue trace) were used to calculate the bar heights at n=12 and 35.