Optimization of Capillary Vibrating Sharp-Edge Spray Ionization for Native Mass Spectrometry of Triplex DNA

Capillary vibrating sharp-edge spray ionization (cVSSI) has been used to study the effects of applied voltage and mass spectrometer heated inlet transfer tube temperature on DNA triplex ion production for native mass spectrometry (MS) samples. Overall, medium applied voltage (-900 to -1000 V) results in better ion production of the desired triplex ions (Tri) (i.e., those without cation adducts such as NH₄ ⁺, Na⁺, and K⁺); mass spectral peak intensities for the [Tri]^8-, [Tri]^9-, and [Tri]^10- ions increase by ∼70, ∼260, and ∼125 fold, respectively, compared to higher voltages (-1100 to -1500 V). The latter voltages result in increased triplex adduct ion (Tri + ad) formation; for the 8-, 9-, and 10- charge states; the ratios of Tri to Tri+ad ion abundances increase by ∼6 fold for the lower voltage. By capillary inlet temperatures of 300 to 400 °C, Tri ion abundances reach maximum values of 6.1 × 10⁵ ([Tri]^8-), 2.9 × 10⁶ ([Tri]^9-), and 6.4 × 10⁵ ([Tri]^10-). Ion abundances for the respective species decrease by ∼4, ∼14, and ∼190 fold at a heated inlet transfer tube temperature of 450 °C. The abundances for Tri+ad ions species generally follow a similar trend as a function of heated inlet transfer tube temperature with the exception that maximum values are obtained at 250 °C. The abundances for DNA triplex fragment ions (Tri-fr) reach maximum values at 400 °C resulting from excessive, in-source ion activation. From these studies, the optimal capillary MS inlet temperature for production of large oligonucleotides by cVSSI is 300 to 350 °C and the applied voltage should be maintained at ∼ -900 V. These studies lay the foundation for native MS of large oligonucleotide species in negative-ion mode exploiting the sensitivity enhancements of cVSSI.