Avoiding H/D Scrambling with Minimal Ion Transmission Loss for HDX-MS/MS-ETD Analysis on a High-Resolution Q-TOF Mass Spectrometer

Abstract

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) enables the study of protein dynamics by measuring the time-resolved deuterium incorporation into a protein incubated in D2O. By using electron-based fragmentation in the gas-phase it is possible to measure deuterium uptake at single residue resolution. However, a prerequisite for this approach is that the solution phase labeling is conserved in the gas-phase prior to precursor fragmentation. It is therefore essential to reduce or even avoid intramolecular hydrogen/deuterium migration, which causes randomization of the deuterium labels along the peptide (hydrogen scrambling). Here we describe an optimization strategy for reducing scrambling to a negligible level while minimizing the impact on sensitivity on a high-resolution Q-TOF equipped with ETD and an electrospray ionization interface consisting of a glass transfer-capillary followed by a dual ion-funnel. In our strategy we have narrowed down the optimization to two accelerating potentials and we have defined the optimization of these in a simple rule by accounting for their interdependency in relation to scrambling and transmission efficiency. Using this rule, we were able to reduce scrambling from 75% to below 5% on average using the highly scrambling-sensitive quadruply charged P1 peptide scrambling probe resulting in a minor 33% transmission loss. To demonstrate the applicability of this approach, we probe the dynamics of certain regions in cytochrome c.

Ultraviolet Photodissociation of Protonated Peptides and Proteins Can Proceed with H/D Scrambling

“…9,[14][15][16][17][18][19][20] On the other hand, electron-based fragmentation methods, in which there is no or negligible vibrational excitation of the precursor ion prior to its dissociation, such as ECD and ETD, do not induce H/D scrambling. 6,10,12,[21][22][23][24][25][26][27][28][29][30] However, any instrumental setting that causes extensive vibrational excitation of the precursor ion will result in H/D scrambling, e.g., harsh desolvation conditions in the ion source. It is therefore critical that the ion source and other instrumental settings are optimized to avoid the occurrence of H/D scrambling in the mass spectrometer.…”

Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

“…This setup is usually able to secure the demanded resolution and sensitivity and is suitable for online coupling to liquid chromatography. Alternatively, one may use a matrix-assisted laser desorption/ionization (MALDI) ion source and QTOF analyzer, since this approach will not produce hydrogen/deuterium scrambling; nevertheless, this setup has the disadvantages of more cumbersome operation and the demand of specific instrumentation for off-line separation and spotting [69][70][71].…”

Gas-phase ion/ion chemistry for structurally sensitive probes of gaseous protein ion structure: Electrostatic and electrostatic to covalent cross-linking

“…64 Hydrogen-deuterium scrambling has also been observed in CID of hydrogen-deuterium exchange mass spectrometry, whereas using rapid electronic activation methods results in the avoidance of label scrambling. 65 Thus, ECD was used to avoid mobilizing the reagents and scrambling the electrostatic reagents' attachment sites.…”