Factors that Promote ESI Multiple Charging for Proteins

摘要

Increasing the charge carried by analytes is desirable because higher charged molecules are more effectively dissociated by tandem MS and because they reduce analyzer m/z requirements. This feature is especially beneficial for the analysis of large, noncovalent complexes. Previously, we demonstrated that multiple charging of native proteins and noncovalent protein complexes could be increased in ESI-MS when spectra are obtained from non-denaturing protein solutions (e.g., 10-50 mM ammonium acetate, pH 6.8) containing up to 0.5percent (v/v) m-nitrobenzyl alcohol (m-NBA) [1]; charge increased by +8percent to +48percent for the native proteins measured. More recently, we presented other compounds as potent as m-NBA (e.g., sulfolane and 3-(trifluoromethyl)-benzyl alcohol) for supercharging under both native and denaturing conditions [2]. The mechanism by which these compounds can enhance ESI charging has not been elucidated fully. Williams and coworkers had previously reported on the "supercharging" phenomenon for compounds such as m-NBA in protein solutions containing methanol/water/acetic acid [3]. Supercharging was attributed to the high relative surface tension of the reagents. However, this mechanism would also predict that m-NBA would not be able to increase protein charging from aqueous solutions. To explain the increasing charge observed for m-NBA, sulfolane, and other compounds in aqueous solution [2], the possibility of protein denaturation (either in solution or in the gas phase) induced by these compounds was raised [4].