Native Ion Mobility–MS Reveals the Bead Ejection Mechanism in Multi-Domain Nucleic Acids

Elucidating how biomolecules acquire charge and retain their solution conformation during electrospray ionization (ESI) is crucial for native MS analysis. 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. The ion mobility results for intermediate charge states are compatible with ion production via the recently proposed bead ejection mechanism (BEM), in addition to the charge residue model (CRM) for low charge states and chain ejection model (CEM) for the highest charge states or for sequences with thymine overhangs on both ends. The position of the G-quadruplex domain and ionic strength governs the charging and retention of G-quadruplex folded regions. Our findings broaden the existing mechanistic framework underpinning nucleic acid ionization.