What Determines the Charge State Distribution of Proteins Electrosprayed from Non-Denaturing Solutions?

Abstract The maximum charge on globular electrosprayed protein ions has been rationalized as the maximal charge sustainable by a solvent drop of comparable size, in turn determined by either small ion evaporation or Rayleigh’s limiting charge. Here we show that spontaneous ion evaporation may continue after the protein ion has dried. Multiply charged cations C+z of bovine serum albumin (BSA) or ovalbumin are electrosprayed into dry ambient air from aqueous solutions of ammonium acetate or thriethylammonium formate. A unique dry protein charge state z is isolated (<1 ms) in a first mobility instrument and passed through a parabolic flow tubular reactor (∼200 ms) where dry ion evaporation transitions C+z → C+z−1 → C+z−2... take place. The protein charge state distribution exiting the reactor is then determined (<1 ms) in a second mobility device. All naturally charged BSA+z and ovalbumin+z ions undergo substantial room temperature charge loss within the reactor, both in ammonium acetate and in triethylammonium formate buffers. The observed maximal charge and the charge distribution are therefore determined by the kinetics of dry ion evaporation from the surface of the dry protein, and depend on the residence time, the temperature and the nature of the evaporating ion. The often-observed low charge states characteristic of buffers containing certain amines is due to the weaker binding of these protonated amines to the protein compared to H+ or NH4+. Maximal charge states may hence be obtained from cold sources with short residence times and selected buffers. The dry ion evaporation here observed may not apply to very large proteins.