Hydrogen-deuterium exchange mass spectrometry (HDX/MS) has become a standard tool for studying the structure and dynamics of proteins, and in recent years it has been successfully applied to DNA oligonucleotides as well. However, the classical approach has a fundamental limitation: if several conformations with the same mass coexist in solution, the measured exchange kinetics represent an average weighted by their relative abundance. This is especially relevant for G-quadruplexes (G4), noncanonical DNA structures that often form complex mixtures of conformers in dynamic equilibrium. Earlier, the authors' group showed that HDX/MS can distinguish ensembles with different numbers of guanine tetrads through differences in cation binding stoichiometry, but conformers with the same number of tetrads remained indistinguishable. In this work, the authors add a third dimension, ion mobility (IMS), which separates ions by their shape and charge, paving the way to conformation-specific kinetics.
The experiments were performed on an Agilent DT-IMS Q-TOF 6560 instrument, where ions pass through a drift tube filled with helium under a uniform electric field. The arrival time of an ion depends on its collision cross section (CCS) and charge, which makes it possible to separate conformations with the same mass-to-charge ratio (m/z). For hydrogen-deuterium exchange, a previously developed continuous-flow scheme was used: a syringe with the sample and a syringe with the exchange buffer were connected through a mixing tee, which allowed different exchange time points to be obtained. After separation by m/z and by arrival time distribution (ATD), exchange curves (the dependence of the number of unexchanged sites on time) were constructed for each population. To separate overlapping ATD peaks, deconvolution with Gaussian functions was applied. In some cases, the fragmentor voltage was increased to improve resolution, but kept below the threshold for gas-phase unfolding. To interpret bimodal isotope distributions, the OligoR software was used, which allows the contributions of EX1 and EX2 exchange mechanisms to be separated.
The authors demonstrated three scenarios for applying HDX/IMS-MS. In the first case, for the telomeric sequence 23TAG, which forms a mixture of hybrid-1 and hybrid-2 conformations with the same mass, they managed to separate them by drift time and measure individual exchange rates. It turned out that hybrid-2 exchanges faster than hybrid-1, and the difference is due not to local fluctuations but to a higher rate of partial unfolding (EX1 mechanism): k_op ≈ 1.76 min⁻¹ versus 0.57 min⁻¹. For the 5YEY sequence, they managed to separate three populations with different CCS values and show that one of them exchanges exclusively via the EX2 mechanism (without unfolding), while the other two show an EX1 contribution with different rates. In the second scenario, using 222T as an example, they showed that IMS allows nonspecifically bound potassium cations, which have the same mass but a different shape, to be filtered out. This made it possible to obtain "clean" kinetics for the specific 2T·1K⁺ and 3T·2K⁺ complexes, excluding the contribution of rapidly exchanging unstructured forms. In the third scenario, they found that for monomorphic G4s (for example, T30177TT) two peaks appear in the ATD, but the exchange rates for them are identical, which points to gas-phase rearrangements of the same structure from solution rather than to different conformations in solution.
The work substantially expands the capabilities of HDX/MS for studying nucleic acids, making it possible to overcome the fundamental limitation associated with averaging over conformations of the same mass. Unlike previous studies, where IMS was used separately for structural analysis or HDX/MS for averaged kinetics, here for the first time both methods are systematically combined to obtain conformation-specific exchange rates. This paves the way for detailed studies of the folding and unfolding mechanisms of DNA structures, as well as for the analysis of complex mixtures that were previously inaccessible. In addition, the work demonstrates that IMS can serve as a filter for removing artifacts of nonspecific binding, which improves the accuracy of HDX/MS data interpretation in general.
The method can be extended to other classes of nucleic acids, including RNA and modified oligonucleotides, as well as to complexes of nucleic acids with proteins or ligands. Further improvement of IMS resolution (for example, with the help of cyclotron ion mobility or structures for lossless ion manipulations) will make it possible to separate even more closely related conformations. Automation of data analysis and the development of algorithms for multidimensional deconvolution will make the method more accessible to a broad range of researchers. Combining it with other methods, such as ion spectroscopy or tandem mass spectrometry with activation, is also promising for obtaining additional structural information.
The method will have an impact on the structural biology of nucleic acids, especially in the study of G-quadruplexes, which are targets for anticancer drugs. It will also be useful in the development of therapeutic oligonucleotides and DNA-based nanostructures.
Apply the method to a broader set of sequences, including RNA quadruplexes and disease-associated mutant forms. Investigate the influence of various cations and ligands on conformational dynamics with resolution down to individual conformers.
In the field of structural biology of nucleic acids, the problem of determining the complete conformational landscape of G-quadruplexes under physiological conditions remains unsolved. This method brings the solution closer by making it possible to link gas-phase observations to behavior in solution and to reveal hidden intermediate states.
Guanine quadruplexes can form structures reminiscent of a stack of pancakes: flat guanine tetrads pile on top of one another, and potassium ions sit between them like filling.
The idea of separating molecules by shape in the gas phase is reminiscent of a fictional "sorter" from a Philip K. Dick story, where objects are classified by nonobvious features. Only here, instead of a fantastic device, there is a drift tube filled with helium.
Not published on arXiv. Here are its page, the PDF and the formalization: you can check it yourself, not only read our account.