Top-Down Structural\nCharacterization of Native Ubiquitin\nCombining Solution-Stable Isotope Labeling, Trapped Ion Mobility Spectrometry,\nand Tandem Electron Capture Dissociation Mass Spectrometry

Solution-phase hydrogen/deuterium exchange (HDX) coupled\nto native\nion mobility spectrometry mass spectrometry (IMS-MS) can provide complementary\nstructural information about the conformational dynamics of biological\nmolecules. In the present work, the solution-stable isotope labeling\n(SIL) combined with trapped ion mobility spectrometry (TIMS) in tandem\nwith top-down electron capture dissociation (ECD) is illustrated for\nthe structural characterization of the solution native states of ubiquitin.\nFour different ubiquitin electrospray solution conditions: (i) single-tip\nnondeuterated, (ii) theta tip for online SIL HDX, (iii) single-tip\nSIL-deuterated, and (iv) theta tip for online SIL H/D back exchange\n(HDbX), were investigated to assess the H/D exchange reactivities\nof native ubiquitin. The combination of TIMS and ECD in a q-ToF MS\ninstrument allowed for additional inspection of gas-phase HDbX added\nby top-down fragmentation, revealing the exposed and protected residues\nwith limited scrambling effects (e.g., intramolecular H/D migration).\nA native charge state distribution (5+ to 7+) and TIMS profiles were\nobserved under the single-tip nondeuterated solution conditions. Mass\nshift distributions of ∼40, ∼104, and ∼87D were\nobserved when incorporating deuterium for online SIL HDX, SIL HDX,\nand online SIL HDbX, respectively, while retaining similar conformational\nstates. ECD fragmentation allowed for the localization of the deuterated\nlabeled residues of the peptide fragments, with a sequence coverage\nof ∼90%, for each of the ubiquitin solution condition. Changes\nin the TIMS trapping time settings (∼70 to ∼795 ms)\nwere used to determine the H/D back exchange dynamics of native ubiquitin.\nHDbX-TIMS-q-ECD-MS/MS exhibited H/D back exchanges in the six-residue\nC-terminal tail as well as around Lys6, Lys11, Lys33, Lys48, and Lys63\nresidues, indicating that these regions are the most exposed area\n(less protected hydrogens) of ubiquitin as compared to the rest of\nthe core residues that adopt a compact β-grasp fold (protected\nhydrogens), which was consistent with the accessible surface area\nof ubiquitin. The present data highlight for the first time consistency\nbetween the solution HDX and gas-phase HDbX-TIMS data for native studies.