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 (UVPD) has recently been introduced as an ion activation method for the determination of single-residue deuterium levels in H/D exchange tandem mass spectrometry experiments. In this regard, it is crucial to know which fragment ion types that can be utilized for this purpose. UVPD yields rich product ion spectra where all possible backbone fragment ion types (a/x, b/y and c/z) are typically observed. Here we provide a detailed investigation of the level of H/D scrambling for all fragment ion types upon UVPD of the peptide scrambling probe P1 (HHHHHHIIKIIK) using an Orbitrap tribrid mass spectrometer equipped with a solid-state 213 nm UV laser. The most abundant UVPD-generated fragment ions (i.e., b/y ions) exhibit extensive H/D scrambling. Similarly, a/x and c/z ions have also undergone H/D scrambling due to UV-induced heating of the precursor ion population. Therefore, dominant b/y ions upon UVPD of protonated peptides is a strong indicator for the occurrence of extensive H/D scrambling of the precursor ion population. In contrast to peptide P1, UV-irradiation of ubiquitin did not induce H/D scrambling in the non-fragmented precursor ion population. However, the UVPD-generated b2 and a4 ions from ubiquitin exhibit extensive H/D scrambling. To minimize H/D scrambling, short UV-irradiation time and high gas pressures are recommended.

No abstract