Affecting Protein Charge State Distributions in Nano-Electrospray Ionization via In-Spray Solution Mixing Using Theta Capillaries

Borosilicate theta glass capillaries pulled to serve as nano-electrospray ionization emitters are used for short time-scale mixing of protein and acid solutions during the electrospray process to alter protein charge state distributions (CSDs) without modifying the sample solution. The extent of protein CSD shifting/denaturing can be tailored by acid identity and concentration. The observed CSD(s) are protein dependent, and the short mixing time-scale enables the study of short-lived unfolding intermediates and higher charge states of noncovalent protein complexes, including those of holomyoglobin. Additionally, the theta tips provide a simple and inexpensive method for mixing nonvolatile reagents such as supercharging agents, which cannot be used with previously developed vapor leak-in techniques, with protein solutions during the electrospray process.