Oligonucleotide charge states in negative ionization electrospray-mass spectrometry are a function of solution ammonium ion concentration

The charge state distribution for oligonucleotides detected using negative ionization electro-spray-mass spectrometry has been studied as a function of solution counterion concentration. In the absence of added buffer, an average charge state (Z) of −7.2 is observed for a 10 μM aqueous solution of a 14mer DNA at pH 7.0, with [M − 8H] 8− the most abundant ion. As the solution concentration of ammonium acetate increases from 0.1 to 33 mM, Z shifts to −3.8 with [M − 4H] 4− the most abundant charge state. The shift in most abundant charge state from [M − 7H] 7− to [M − 4H] 4− occurs abruptly between 1.0 and 10 mM NH 4OAc. Above 100 mM NH 4OAc, the value of Z plateaus at −3.1, with [M − 3H] 3− the most abundant charge state. The addition of 1–50 mM glycine to the analyte solution does not alter Z, suggesting that the changes in charge state observed by using ammonium acetate result from a solution equilibration of cations around the DNA strand, rather than nonspecific gas-phase proton transfers during the electrospray process. The fraction of neutralized phosphate groups reaches a maximum of 0.79 ± 0.03 independent of length and sequence.