Valérie Gabelica

Hydrogen/deuterium exchange coupled to mass spectrometry (HDX/MS) is a powerful technique to probe nucleic acid secondary structures and dynamics, but its ability to resolve conformers with identical masses remains limited. To overcome this challenge, we integrated ion mobility spectrometry (IMS) into our native HDX/MS workflow, and tested the approach on a variety of model DNA G-quadruplex structures. We show several examples of human telomeric sequence oligonucleotides where complexes of the same mass differ in their collision cross section, and each gas-phase conformational ensembles corresponds to unique solution exchange behaviors, allowing kinetic analysis beyond what is possible with HDX/native MS alone. But we also found examples where several gas-phase populations separated in ion mobility have exactly the same solution exchange behavior, suggesting that conformational rearrangements occur either during electrospray or at later stages in the gas phase. Finally, we show how IMS filtering can be leveraged to distinguish groups of non-specific cation binding on a given conformational ensemble, as indicated by populations with different masses and same exchange rates. These findings establish IMS as an essential tool for complementing HDX/MS in the characterization of structural polymorphism and conformational ensembles in DNA oligonucleotides.

HIVpro1 sequence is derived from HIV-1 promoter. It forms a non-canonical DNA structure called G-quadruplex (or G4) that is assembled trough the stacking of two guanine tetrads. DBA1 ligand binds to the external tetrad of HIVpro1 G4.

We describe here various ion activation experiments realized in the Omnitrap platform integrated on the timsOmni mass spectrometer for the analysis of oligonucleotides in the negative ion mode. The activation methods include resonance collision-induced dissociation (_RCID), electron detachment dissociation (EDD), infrared laser multiple-photon activation (IRMPD) and UV laser photodissociation (UVPD) at 266 nm. Special emphasis is given to EDD, either as a standalone technique or in conjunction with vibrational reactivation of the ion radicals. We describe EDD on standard 6-mer DNA sequences that have been extensively characterized on other instruments, followed by a comparison of several activation approaches for the phosphorothioate-based oligonucleotide therapeutics Fomivirsen, and conclude with the fragmentation analysis of 46-mer DNA and RNA. EDD alone already provides excellent sequence information on short oligonucleotides such as Fomivirsen, but MS³ combinations such as EDD-_RCID or EDD-IRMPD proved even more effective, including for the 46-mer DNA (less prone to fragmentation than RNA) at a relatively low charge states. The diversity of ion activation combinations available on the Omnitrap platform is demonstrated by MS⁴ experiments, which experimentally validate the formation of d ions from a• radical fragments, and w ions from z• radical fragments produced by EDD.

Understanding how biomolecules acquire their charge and retain their solution conformation during electrospray ionization (ESI) is crucial for native mass spectrometry (native MS) interpretation. Here, we examine the charging and gas phase conformation of nucleic acid constructs comprising folded G-quadruplex "beads" linked by unstructured polythymine regions. Under physiological ionic strength, these oligonucleotides exhibit a multimodal charge-state and collision cross section distribution, revealing multiple conformational ensembles, in contrast to the unimodal profiles typically observed for shorter oligonucleotides. Native MS observations for intermediate charge states are compatible with ion production via the recently proposed bead-ejection scenario, in addition to the charge residue scenario for low charge states and chain ejection for the highest charge states or for sequences with thymine overhangs on both ends. The preservation of the local structures in ions charged above the Rayleigh limit helps to infer the presence of folded subunits. The position of the G-quadruplex subunit and ionic strength govern the charging and retention of G-quadruplex folded regions. Our findings broaden the existing conceptual framework underpinning nucleic acid ionization.

We investigated the G-quadruplex (G4) binding selectivity of short aromatic oligoamide helical foldamers comprising quinoline (Q) and pyridine (P) units. We found that the foldamers bind with 1:1 and 2:1 stoichiometries and prefer parallel G4 structures, especially when the external G-quartets are sterically accessible. A crystal structure of the tetramer QQPQ with the parallel G4 formed by dTGGGTTGGGTTGGGTTGGGT shows two quinoline subunits interacting with an external G-quartet through π-stacking, and solution nuclear magnetic resonance (NMR) confirms that the foldamer targets the 3′ and 5′ ends of this G4. Foldamers can also selectively target sequence variants of the telomeric sequences containing adenine-to-thymine mutation in the loops. The conformational selectivity of foldamers originates from the bulkiness of oligomers with four or more subunits, which imposes steric restrictions on G4 binding. The flexibility provided by the pyridine subunits was also key to improve affinity. Mixed quinoline–pyridine foldamers are thus a promising class of selective G4 ligands, and their unique modular scaffold offers new avenues to further improve their affinity and selectivity.

We describe here various ion activation experiments realized in the Omnitrap™ platform integrated on the timsOmni ^TM mass spectrometer for the analysis of oligonucleotides in the negative ion mode. The activation methods include resonance collision-induced dissociation ( _R CID), electron detachment dissociation (EDD), infrared laser multiple-photon activation (IRMPD) and UV laser photodissociation (UVPD). Special emphasis is given to EDD, either as a standalone technique or in conjunction with vibrational re-activation of the ion radicals. We describe EDD on standard 6-mer DNA sequences that have been extensively characterized on other instruments, followed by a comparison of several activation approaches for the phosphorothioate-based oligonucleotide therapeutics Fomivirsen, and concluding with the fragmentation analysis of 46-mer DNA and RNA. EDD alone already provides excellent sequence information on Fomivirsen, but MS ³ combinations such as EDD- _R CID or EDD-IRMPD proved even more effective, including for the 46-mer DNA (less prone to fragmentation than RNA) at a relatively low charge state. The diversity of ion activation combinations available on the Omnitrap platform is demonstrated by an MS ⁴ experiment investigating the fate of a • and z • radical fragments produced by EDD. TOC graphics:

Understanding how biomolecules acquire their charge and retain their solution conformation during electrospray ionization (ESI) is crucial for native mass spectrometry (native MS) interpretation. Here, we examine the charging and gas phase conformation of nucleic acid constructs comprising folded G-quadruplex “beads” linked by unstructured polythymine regions. Under physiological ionic strength, these oligonucleotides exhibit a multimodal charge-state and collision cross section distribution, revealing multiple conformational ensembles, in contrast to the unimodal profiles typically observed for shorter oligonucleotides. Native MS observations for intermediate charge states are compatible with ion production via the recently proposed bead ejection scenario, in addition to the charge residue scenario for low charge states and chain ejection for the highest charge states or for sequences with thymine overhangs on both ends. The preservation of the local structures in ions charged above the Rayleigh limit helps to infer the presence of folded subunits. The position of the G-quadruplex subunit and ionic strength governs the charging and retention of G-quadruplex folded regions. Our findings broaden the existing conceptual framework underpinning nucleic acid ionization. TOC graphic:

We investigated the G-quadruplex binding selectivity of short aromatic oligoamide helical foldamers comprising quinoline (Q) and pyridine (P) units. We found that the foldamers prefer parallel G-quadruplex structures, especially when the external G-quartets are sterically accessible. A crystal structure of the tetramer QQPQ with the parallel G-quadruplex formed by dTGGGTTGGGTTGGGTTGGGT shows two quinoline subunits interacting with an external G-quartet through π-stacking, and solution NMR confirms that the foldamer targets the 3’ and 5’ ends of this G-quadruplex. Foldamers can also selectively target sequence variants of the telomeric sequences containing adenine-to-thymine mutation in the loops. The conformational selectivity of foldamers originates from the bulkiness of oligomers with four or more subunits, which imposes steric restrictions on G-quadruplex binding. The flexibility provided by the pyridine subunits was also key to improve affinity. Mixed quinoline-pyridine foldamers are thus a promising class of selective G-quadruplex ligands, and their unique modular scaffold offers new avenues to further improve their affinity and selectivity.

Ionization and fragmentation are at the core of mass spectrometry. But they are not necessarily separated in space, as in‐source fragmentation can also occur. Here, we survey the literature published since our 2005 review on the internal energy and fragmentation in electrospray ionization sources. We present new thermometer molecules to diagnose and quantify source heating, provide tables of recommended threshold ( E ₀ ) and appearance energies ( E _app ) for the survival yield method, and attempt to compare the softness of a variety of ambient pressure ionization sources. The droplet size distribution and desolvation dynamics play a major role: lower average internal energies are obtained when the ions remain protected by a solvation shell and spend less time nakedly exposed to activating conditions in the transfer interface. Methods based on small droplet formation without charging can thus be softer than electrospray. New dielectric barrier discharge sources can gas‐phase ionize small molecules while conferring barely more internal energy than electrospray ionization. However, the tuning of the entire source interface often has an even greater influence on ion internal energies and fragmentation than on the ionization process itself. We hope that this review will facilitate further research to control and standardize in‐source ion activation conditions, and to ensure the transferability of data and research results in mass spectrometry.