Supercharging of Proteins by Salts during Polarity Reversed Nano-Electrospray Ionization.

Supercharging is beneficial in many ways to the analysis of proteins by mass spectrometry (MS). In this work, a novel supercharging method was developed. It made use of our previously developed ionization technique: namely, polarity reversed nanoelectrospray ionization (PR-nESI) for the ionization of proteins. Supercharging of proteins was achieved by just adding 1-10 mM of a salt to the sample, such as sodium chloride (NaCl). The charge state of proteins obtained by our method was significantly higher than that by nano-ESI with 1% (v/v) acetic acid (HAc). Different kinds of salts were investigated. Salts with  strong acid anions were capable of supercharging proteins, including chlorides, bromides, iodides, and nitrates. The signal intensity and signal to noise ratio ( S/ N) of proteins were increased at the same time. Phosphates were also found to have a supercharging effect, due to the fact that phosphoric acid was a medium-strong acid. In comparison, salts with weak acid anions had no supercharging effect, such as carbonates, sulfides, acetates, and formates. The species of the salt anion was critical to the supercharging effect, while the species of the salt cation showed little influence on the supercharging effect. Investigations were made into the mechanism of our method. The supercharging effect was caused by interactions between protein molecules and salt anions, as well as the influence of protons. The present work offered us an alternative way for the supercharging of proteins. The use of common salts for supercharging made the procedure more convenient. The concentration of salts needed for supercharging was much lower than those conventionally used for supercharging reagents. Taking into consideration the fact that many biological samples are buffered with phosphates and chlorides, these samples could be directly supercharged by our method without any additional additives. Furthermore, as many salts are nontoxic and can easily be found in a chemical laboratory, the use of salts for supercharging would be a much more practical and economical choice. In addition, the present work also furthered our understandings about the mechanism of supercharging, as well as electrospray.