Mobility Analysis of Proteins by Charge Reduction in a Bipolar Electrospray Source.

Analysis of large electrosprayed biopolymers by electrical mobility alone is greatly facilitated by reducing their charge to unity. Here, we combine within a single chamber positive aqueous electrospray (ES), producing multiply charged protein cations, with negative methanolic ES, yielding small singly charged anions. Use of a 100 mM triethylammonium formate buffer in both solutions yields very small drops. The two sprays are decoupled electrostatically by an interposed 50% transparent, grounded metallic grid. This screen is readily crossed by the ions, resulting in substantial charge reduction. In spite of the grid, the aqueous spray is easily destabilized by the presence of anions in the positive ES region. Nonetheless, practical ES stabilization is achieved by using relatively small capillary tips (∼15 μm) in the positive emitter. Protein peaks obtained are as narrow as those previously reported via charge reduction with a radioactive Ni-63 source. Controlling the position of the negative ES permits spanning the full range of charge states, from high natural values to predominantly singly charged ions, even for large proteins such as immunoglobulin G (∼150 kDa).