Concomitant species that appear at the same or very similar times in a mass-spectral analysis can clutter a spectrum because of the coexistence of many analyte-related ions (e.g., molecular ions, adducts, fragments). One method to extract ions stemming from the same origin is to exploit the chemical information encoded in the time domain, where the individual temporal appearances inside the complex structures of chronograms or chromatograms differ with respect to analytes. By grouping ions with very similar or identical time-domain structures, single-component mass spectra can be reconstructed, which are much easier to interpret and are library-searchable. While many other approaches address similar objectives through the Pearson’s correlation coefficient, we explore an alternative method based on a modified cross-correlation algorithm to compute a metric that describes the degree of similarity between features inside any two ion chronograms. Furthermore, an automatic workflow was devised to be capable of categorizing thousands of mass-spectral peaks into different groups within a few seconds. This approach was tested with direct mass-spectrometric analyses as well as with a simple, fast, and poorly resolved LC–MS analysis. Single-component mass spectra were extracted in both cases and were identified based on accurate mass and a mass-spectral library search.

Although allosteric binding of small molecules is commonplace in protein structures, it is rather rare in DNA species such as G-quadruplexes. By using CD melting, here, we found binding of the small-molecule ligands PDS and L2H2-6OTD to the telomeric DNA G-quadruplex was cooperative. Mass spectrometry indicated a 1:1:1 ratio in the ternary binding complex of telomeric G-quadruplex, PDS, and L2H2-6OTD. Compared to the binding of each individual ligand to the G-quadruplex, single-molecule mechanical unfolding assays revealed a significantly decreased dissociation constant when one ligand is evaluated in presence of another. This demonstrates that cooperative binding of PDS and L2H2-6OTD to the G-quadruplex is allosteric, which is also supported by the mass spectra data that indicated the ejection of coordinated sodium ions upon binding of the hetero-ligands to the G-quadruplex. The unprecedented observation of the allosteric ligand binding to higher ordered structures of DNA may help to design more effective ligands to target non-B DNA species involved in many critical cellular processes.