Ionic strength of electrospray droplets affects charging of DNA oligonucleotides

The fundamental aspects of charging in electrospray ionization (ESI) are hotly debated. In the present study, ESI charging of DNA oligonucleotides was explored in both positive (ESI+) and negative (ESI-) polarity using mass spectrometry detection. Single-stranded 12-mer CCCCAATTCCCC in buffer solution (aqueous NH4Ac, 100 mM) produced similar charge state distribution (CSD) in either ESI+ or ESI-. Similarity of CSD in ESI+ and ESI- was also observed for the double-stranded 12-mer CGCGAATTCGCG. By adding typical low-vapor reagents (e.g. m-nitro benzyl alcohol, m-NBA; sulfolane) into the same buffer solution (<0.5% w/v), both CCCCAATTCCCC and CGCGAATTCGCG revealed strong supercharging (SC) effect in ESI-, while very little or no SC effect was observed in ESI+. With either sulfolane or m-NBA, the CGCGAATTCGCG duplex dissociated into single strands in ESI-. No SC was observed in both ESI+ and ESI- for thermally denatured CGCGAATTCGCG duplex in NH4 Ac buffer without the reagents. These findings are difficult to reconcile with the earlier model, which attributes SC in aqueous buffer solution to the conformational changes of analytes. Our observations suggest that the ionic strength of ESI droplets strongly affects the CSD of biopolymers such as DNA oligonucleotides and that SC effect is related to the depletion of ionic strength during the ESI process.