Strong Acid Anions Significantly Increasing the Charge State of Proteins during Electrospray Ionization

Regulation of protein's charge state in electrospray is of great importance to the analysis of proteins. Different methods have been developed so far to increase the charge state of proteins. In this work, we investigated the influence of different anions on the charge state of proteins. Both strong acid anions and weak acid anions were taken into consideration. The results showed that the presence of 5 mM strong acid anions in acidic solutions could significantly increase the charge state of proteins. In comparison, weak acid anions with the same concentration in solution had little impact on the charge state of proteins. The species of the cations in the samples had very limited influence on the charge state. The presence of a certain amount of acid in sample solution was critical to the effect of strong acid anions. Almost no increase of the charge state was observed when no acid was added to the samples. However, remarkable increase of the charge state of myoglobin (Mb) was observed when 0.001% (v/v) acetic acid (HAc) was added to the sample together with 5 mM sodium chloride (NaCl). A higher concentration of acid in samples would further enhance the effect of strong acid anions on the increase of the charge state. Further investigations into the mechanism revealed that the effect of the strong acid anions on the charge state of proteins was based on the unfolding of the protein molecules during electrospray ionization (ESI). The interactions among H⁺, anions, and protein molecules were so strong that it caused the unfolding of protein molecules and resulted in the increasing of proteins' charge states. The key factor that made strong acid anions and weak acid anions different in the results was the hydrolysis of the weak acid anions in acidic solutions. The present work furthers our understanding about electrospray, as well as the regulation of protein charge state. The presence of strong acid anions in acidic solutions can significantly influence the charge state of proteins in electrospray. Attention should be paid to this when regulating the charge state of proteins.