Charge State Manipulation of Proteins using Continuous Flow - Extractive Desorption Electrospray Ionization (CF-EDESI) - Mass Spectrometry

摘要

Experiments with cytochrome c demonstrated the use of CF-EDESI in protein charging analysis. Despite extremely denaturing conditions (10percent HOAc), analysis using CF-EDESI was able to observe a bimodal distribution of cytocrhome c, where the protein is predominantly retained in the folded conformation as seen through comparisons of the charge state distributions of both folded/unfolded forms. Lysozyme was also successfully increased to higher charge states in a similar fashion using sulfolane. Investigations of myoglobin using three different charging additives indicated that the ratio of holoenyzme to apoenyzme was consistent despite increasing concentrations of denaturing additives. Increasing concentrations of these agents still increased the average charge state distribution of both forms of the protein demonstrating the versatility of performing protein charging experiments without directly infusion of charging reagents with proteins prior to analysis. Definitive proof of the capabilities of CF-EDESI to maintain noncovalent interactions of protein complexes requires a greater percentage of myoglobin to be in the holoenzyme form in normal physiological conditions. Further efforts are being made to obtain such conditions beforehand, including a CF-EDESI source incorporating a softer nanospray configuration.