Nanopore Liberates G-Quadruplexes from Biochemical Buffers for Accurate Mass Spectrometric Examination

Achieving accurate measurements of G-quadruplexes (G4s), especially the characterization of their complicated non-covalent interactions with various components (such as metal ions and ligands) under physiological conditions, is of fundamental significance in unveiling their biological roles and developing antitumor drugs. By employing a nanopore ion emitter (∼30 nm), we demonstrated for the first time that G4 ions, which are free of non-specific adduction and meanwhile maintaining their pre-existing specific bindings with metal ions or ligands, can be directly liberated from common biochemical buffers (consisting of concentrated non-volatile salts of >150 mM) for mass spectrometric examination. Notably, the intermediate complexes of G4s with mixed di-cation coordination formed during the Na⁺/K⁺ exchange were successfully observed by mass spectrometry, whose structures were also revealed by the reconstructed circular dichroism spectra. We believe the nanopore-based ion emitters have built a solid bridge between native G4s in aqueous buffers and their accurate stoichiometries obtained by mass spectrometric examination.