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. 2012 Feb;23(2):301-9.
doi: 10.1007/s13361-011-0298-2. Epub 2011 Dec 1.

Automated hydrogen/deuterium exchange electron transfer dissociation high resolution mass spectrometry measured at single-amide resolution

Rachelle R Landgraf  1 , Michael J Chalmers, Patrick R Griffin
Affiliations

Automated hydrogen/deuterium exchange electron transfer dissociation high resolution mass spectrometry measured at single-amide resolution

Rachelle R Landgraf et al. J Am Soc Mass Spectrom. 2012 Feb.

Abstract

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a well established method for the measurement of solution-phase deuterium incorporation into proteins, which can provide insight into protein conformational mobility. However, most HDX measurements are constrained to regions of the protein where pepsin proteolysis allows detection at peptide resolution. Recently, single-amide resolution deuterium incorporation has been achieved by limiting gas-phase scrambling in the mass spectrometer. This was accomplished by employing a combination of soft ionization and desolvation conditions coupled with the radical-driven fragmentation technique electron transfer dissociation (ETD). Here, a hybrid LTQ-Orbitrap XL is systematically evaluated for its utility in providing single-amide deuterium incorporation for differential HDX analysis of a nuclear receptor upon binding small molecule ligands. We are able to show that instrumental parameters can be optimized to minimize scrambling and can be incorporated into an established and fully automated HDX platform making differential single-amide HDX possible for bottom-up analysis of complex systems. We have applied this system to determine differential single amide resolution HDX data for the peroxizome proliferator activated receptor bound with two ligands of interest.

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Figures

Figure 1
Figure 1
(a) Three-dimensional representation of the PPARγ ligand binding domain. The area highlighted in blue is the peptide IRIFQGCQF in helix 3. (b) Structural details of helix 3 peptide. (c) Build up curves illustrating the differences in deuterium incorporation upon binding of rosiglitazone and MRL24 to PPARγ
Figure 2
Figure 2
HDX-ETD-HRMS analysis of PPARγ. (a) Chromatographic separation of PPARγ peptic peptides. (b) Full scan mass spectrum of the peptide [IRIFQGCQF]2+. (c) ETD HRMS/MS mass spectrum of the fragmented peptide [IRIFQGCQF]2+
Figure 3
Figure 3
Gauging gas-phase scrambling through the loss of NH3. The singly-charged reduced species of the IRIFQGCQF peptide and its NH3-deficient counterpart for (a) apo PPARγ, (b) MRL24 bound PPARγ, and (c) rosiglitazone bound PPARγ
Figure 4
Figure 4
Isotopic patterns of the c3–c8 product ions with no deuterium labeling, 60 s on-exchange of apo PPARγ and 60 s on-exchange of the MRL24 and rosiglitazone bound PPARγ. These ETD spectra were acquired with the same “low scrambling” instrument parameters used to generate the data shown in Supplemental Figure 1
Figure 5
Figure 5
Bar charts showing the deuterium content of the c product ions for the peptide IRIFQGCQF2+ for the 10, 30, 60, 300, 900, and 3600 s on-exchange time points. An increase in deuterium content indicates an amide that has incorporated deuterium
Figure 6
Figure 6
Differential HDX of the peptide IRIFQGCQF overlaid on the three-dimensional structure (PDB 2PRG). Protection observed at the peptide level for PPARγ bound to (a) MRL24 and (b) rosiglitazone. Protection observed at the single-amide level for PPARγ bound to (b) MRL24 and (d) rosiglitazone. Helices 10, 11, and 12 have been removed for a clearer view of helix 3
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