Supercharging Proteins to Near the Theoretical Limit via Reduced Pressure Ionization

Electrospray ionization (ESI) is well-known for generating multiply charged protein ions. Higher charge states enhance tandem mass spectrometry (MS/MS) by improving fragmentation efficiency and increasing sequence coverage. However, the extent of protein charging can be limited by many factors including proton transfer reactions with ambient gases. Here, we demonstrate that droplet desolvation under reduced pressure significantly increases protein charge states, especially when combined with chemical supercharging additives such as 1,2-butylene carbonate. Using reduced pressure nanoelectrospray ionization (250 Torr), the maximum observed charge states of cytochrome <i>c</i> (12.6 kDa) and myoglobin (16.7 kDa) increased from 24+ and 32+ (under ambient conditions) to 30+ and 40+, respectively, within 15% of their predicted <i>in vacuo</i> charge state limits. Enhanced charging was also achieved for larger proteins. For example, carbonic anhydrase (29 kDa) and bovine serum albumin (BSA, 66.5 kDa) reached charge states of 62+ and 112+ under reduced pressure, compared to 46+ and 92+ at ambient pressure. These results demonstrate that combining reduced pressure desolvation with chemical supercharging is an effective approach for increasing protein charge states in ESI to near the theoretical limit, and should offer advantages for the top-down analysis of large biomolecules.