cytochrome-c-t and propylene-carbonate

The effectiveness of the supercharging reagents m-nitrobenzyl alcohol (m-NBA) and propylene carbonate at producing highly charged protein ions in electrospray ionization is compared. Addition of 5% m-NBA or 15% propylene carbonate increases the average charge of three proteins by ∼21% or ∼23%, respectively, when these ions are formed from denaturing solutions (water/methanol/acetic acid). These results indicate that both reagents are nearly equally effective at supercharging when used at their optimum concentrations. A narrowing of the charge state distribution occurs with both reagents, although this effect is greater for propylene carbonate. Focusing the ion signal into fewer charge states has the advantage of improving sensitivity. The maximum charge state of ubiquitin formed with propylene carbonate is 21+, four charges higher than previously reported. Up to nearly 30% of all residues in a protein can be charged, and the collisional cross sections of the most highly charged ions of both ubiquitin and cytochrome c formed with these supercharging reagents were measured for the first time and found to be similar to those calculated for theoretical highly extended, linear or near-linear conformations. Under native supercharging conditions, m-NBA is significantly more effective at producing high charge states than propylene carbonate.

Topics: Benzyl Alcohols; Carbonic Anhydrases; Cytochromes c; Ions; Propane; Protein Conformation; Solubility; Tandem Mass Spectrometry; Ubiquitin

The effectiveness of two new supercharging reagents for producing highly charged ions by electrospray ionization (ESI) from aqueous solutions in which proteins have native structures and reactivities were investigated. In aqueous solution, 2-thiophenone and 4-hydroxymethyl-1,3-dioxolan-2-one (HD) at a concentration of 2% by volume can increase the average charge of cytochrome c and myoglobin by up to 163%, resulting in even higher charge states than those that are produced from water/methanol/acid solutions in which these proteins are denatured. The greatest extent of supercharging occurs in pure water, but these supercharging reagents are also highly effective in aqueous solutions containing 200 mM ammonium acetate buffer commonly used in native mass spectrometry (MS). These reagents are less effective supercharging reagents than m-nitrobenzyl alcohol (m-NBA) and propylene carbonate (PC) when ions are formed from water/methanol/acid. The extent to which loss of the heme group from myoglobin occurs is related to the extent of supercharging. Results from guanidine melts of cytochrome c monitored with tryptophan fluorescence show that the supercharging reagents PC, sulfolane and HD are effective chemical denaturants in solution. These results provide additional evidence for the role of protein structural changes in the electrospray droplet as the primary mechanism for supercharging with these reagents in native MS. These results also demonstrate that for at least some proteins, the formation of highly charged ions from native MS is no longer a significant barrier for obtaining structural information using conventional tandem MS methods.

Topics: Acetates; Benzyl Alcohols; Cytochromes c; Indicators and Reagents; Ions; Methanol; Myoglobin; Propane; Protein Conformation; Proteins; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry; Tandem Mass Spectrometry; Thermodynamics; Thiophenes; Water