Combined strategies for suppressing nonspecific cationic adduction to G-quadruplexes in electrospray ionization mass spectrometry.

G-quadruplex secondary structures are naturally found in genome sequences and play essential roles in regulating a wide variety of important biological processes. Although stabilizing effects of monovalent cations (e.g., K+ and Na+) has been recognized during the past decades, a general and reliable analytical method for accurate characterization of specific interactions of K+/Na+ with G-quartets is still not well established. In the present study, we demonstrate a practical strategy that combined the use of a nanoscale ion emitter, a low-flow drying gas and a volatile salt (trimethylammonium acetate) to almost totally suppress the nonspecific cationic adduction to G-quadruplexes during the ionization process. Our combined strategy takes full advantage of the ultrasmall initial charged droplets when employing a nanoscale ion emitter, the maximum uneven fission of charged droplets under the gentle desolvation conditions, and the effective shielding of the negatively charged phosphate groups by trimethylammonium ions, to eventually producing ions of G-quadruplexes free of non-specific K+/Na+ adduction. For the first time, the accurate binding states as well as the quantitative binding constants between K+/Na+ and G-quadruplexes can be directly obtained even in the presence of tens of millimolar non-volatile salts, which has long been a notorious challenge in mass spectrometry. Accurate binding state between Na + and 24TTG directly obtained by electrospray ionization mass spectrometry using a combined strategy for suppressing nonspecific cationic adduction. [Display omitted] • Combined use of a nanoscale ion emitter, a low-flow drying gas, and TMAA to avoid nonspecific cationic adduction in ESI-MS. • Unambiguous characterization of the pre-exist in-solution specific binding stoichiometry between G4s and K+. • Direct characterization of weak interactions between G4s and Na+ even in tens of millimolar non-volatile salts. • Insights into the topological distinctiveness between G4-K+ and G4-Na+ complexes in terms of their unique binding stoichiometry. [ABSTRACT FROM AUTHOR]