Automated Hydrogen/Deuterium Exchange Electron Transfer Dissociation High Resolution Mass Spectrometry Measured at Single-Amide Resolution

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.

Quantitative Protein Topography Analysis and High-Resolution Structure Prediction Using Hydroxyl Radical Labeling and Tandem-Ion Mass Spectrometry (MS)*

“…MS2 based quantification is in principle a general solution to the problem of increasing structural resolution, and has been attempted for HDX-MS, but the scrambling of the labels in the gas phase has been difficult to overcome using collision induced dissociation (15,16). Alternative approaches for HDX-MS site localization, like electron transfer dissociation to achieve single residue resolution have potential promise but are typically limited to larger peptides that can access higher charge states easily (17,18). MS2 strategies to enhance the resolution for covalent labeling experiments have been attempted with some success, as scrambling is not a limitation in covalent labeling experiments (7, 19 -21).…”

Mass spectrometry‐based methods to study protein architecture and dynamics

“…In some cases, this may allow the backbone amide protection patterns to be determined at a single-residue resolution, 69 although such instances remain very rare. Supplementation of enzymatic digestion with peptide ion fragmentation in the gas phase may also enhance the spatial resolution of HDX MS measurements, 64,[70][71][72] although extreme care must be taken to avoid possible gas phase artifacts leading to hydrogen scrambling within the peptide ion prior to its fragmentation. 73 In addition to limited spatial resolution, HDX MS measurements frequently suffer from incomplete sequence coverage, especially when applied to larger and extensively glycosylated proteins.…”

Nepenthesin from Monkey Cups for Hydrogen/Deuterium Exchange Mass Spectrometry

“…Peptides may be detected with more sensitivity than proteins, and samples of considerably higher complexity can be interrogated (11). Analytically, this shifts the focus toward optimizing protein digestion, to cover 100% of the sequence and generate a high degree of overlapping fragments to increase opportunities for localizing the deuterium label at high resolution (12)(13)(14).…”