Multidimensional separations of ubiquitin conformers in the gas phase: relating ion cross sections to H/D exchange measurements

Abstract

Investigating gas-phase structures of protein ions can lead to an improved understanding of intramolecular forces that play an important role in protein folding. Both hydrogen/deuterium (H/D) exchange and ion mobility spectrometry provide insight into the structures and stabilities of different gas-phase conformers, but how best to relate the results from these two methods has been hotly debated. Here, high-field asymmetric waveform ion mobility spectrometry (FAIMS) is combined with Fourier-transform ion cyclotron resonance mass spectrometry (FT/ICR MS) and is used to directly relate ubiquitin ion cross sections and H/D exchange extents. Multiple conformers can be identified using both methods. For the 9+ charge state of ubiquitin, two conformers (or unresolved populations of conformers) that have cross sections differing by 10% are resolved by FAIMS, but only one conformer is apparent using H/D exchange at short times. For the 12+ charge state, two conformers (or conformer populations) have cross sections differing by <1%, yet H/D exchange of these conformers differ significantly (6 versus 25 exchanges). These and other results show that ubiquitin ion collisional cross sections and H/D exchange distributions are not strongly correlated and that factors other than surface accessibility appear to play a significant role in determining rates and extents of H/D exchange. Conformers that are not resolved by one method could be resolved by the other, indicating that these two methods are highly complementary and that more conformations can be resolved with this combination of methods than by either method alone.

Figure 1

Comparison of a CV scan for the 8+ charge state of ubiquitin obtained with a FT/ICR mass spectrometer (line with letters x) with a scan obtained previously by Purves et al. (solid line) [14]. Collisional cross section measurements made by Purves et al. [14] for this charge state are indicated by the dashed line with error bars. Letters a to d indicate CV values for the H/D exchange mass spectra shown in Figure 3.

Figure 2

Compensation voltage scans for the 8+ to 13+ charge state of bovine ubiquitin.

Figure 3

H/D exchange mass spectra for the 8+ charge state of ubiquitin with 20 s reaction time at ~8 × 10⁻⁷ torr at compensation voltages of (a) −4.4 V, (b) −5.3 V, (c) −5.9 V, and (d) −6.5 V corresponding to a 300 Ų range of collisional cross sections.

Figure 4

FAIMS-FT/ICR MS data for the 9+ charge state of ubiquitin: (a) CV scan and (b), (c), (d), (e) H/D exchange measured at CV values of −5.0 V (b), (d) and −5.8 V (c), (e) at 20 s (b), (c) and 40 s exchange time (d), (e).

Figure 5

FAIMS-FT/ICR MS data for the 10+ charge state of bovine ubiquitin measured using 20 s H/D reaction time: (a) surface plot of CV values versus extents of H/D exchange, where lines marked with open triangles and filled triangles indicate H/D exchange distributions at CV values of −4.9 V and −5.5 V, respectively, and lines marked with open circles and letters x correspond to CV scans of the H/D exchange distribution with 7 and 21 deuteriums incorporated, respectively, (b) and (c) H/D exchange mass spectra at −4.9 V (b) and −5.5 V (c) and (d) CV scan with no H/D exchange (solid line) and with H/D exchange of the conformer that undergoes 7 exchanges (open circles) and 21 exchanges (letters x).

Figure 6

FAIMS-FT/ICR MS data for the 11+ charge state of bovine ubiquitin: (a) CV scan and spectra obtained at CV values of (b) −5.0 V and (c) −5.4 V with 40 s reaction time.

Figure 7

FAIMS-FT/ICR MS data for the 12+ charge state of bovine ubiquitin: (a) CV scan with no H/D exchange (solid line), conformer with 17 exchanges (letter x), and conformer with 2 exchanges (open circles) where b, c, d, and e indicate the compensation voltages at which the H/D exchange mass spectra were acquired (20 s reaction time for (b)–(d), no H/D exchange for (e).