Effects of solvent on the maximum charge state and charge state distribution of protein ions produced by electrospray ionization

Abstract

The effects of solvent composition on both the maximum charge states and charge state distributions of analyte ions formed by electrospray ionization were investigated using a quadrupole mass spectrometer. The charge state distributions of cytochrome c and myoglobin, formed from 47%/50%/ 3% water/solvent/acetic acid solutions, shift to lower charge (higher m/z) when the 50% solvent fraction is changed from water to methanol, to acetonitrile, to isopropanol. This is also the order of increasing gas-phase basicities of these solvents, although other physical properties of these solvents may also play a role. The effect is relatively small for these solvents, possibly due to their limited concentration inside the electrospray interface. In contrast, the addition of even small amounts of diethylamine (<0.4%) results in dramatic shifts to lower charge, presumably due to preferential proton transfer from the higher charge state ions to diethylamine. These results clearly show that the maximum charge states and charge state distributions of ions formed by electrospray ionization are influenced by solvents that are more volatile than water. Addition of even small amounts of two solvents that are less volatile than water, ethylene glycol and 2-methoxyethanol, also results in preferential deprotonation of higher charge state ions of small peptides, but these solvents actually produce an enhancement in the higher charge state ions for both cytochrome c and myoglobin. For instruments that have capabilities that improve with lower m/z, this effect could be taken advantage of to improve the performance of an analysis.Electrospray ionization (ESI) [1] is well recognized as a soft ionization method for producing gas-phase ions of large biopolymers, such as oligonucleotides, proteins, and even noncovalent biomolecular complexes [2]. In combination with mass spectrometry, molecular masses of large molecules can be measured with unprecedented accuracy [3]. The multiple charging of large analyte ions that occurs with ESI has the advantage that the masses of very large molecules can be measured using mass spectrometers with upper mass-to-charge limits. One outstanding challenge in ESI mass spectrometry is to accurately predict the observed charge state distribution of a large molecule, given its primary structure, the composition of the solvent system from which the ions are formed, instrumental conditions, etc. Several factors have been shown to influence the observed charge state distribution, including molecular conformation [4][5][6], acid-base chemistry both in solution and in the gas phase [7][8][9][10][11], solvent composition [8], instrumental factors, etc. Several models have been proposed to qualitatively account for some of these effects [12][13][14][15]. A general conclusion from several studies is that the electrospray charge state distributions of proteins formed from denaturing solution conditions are shifted to higher charge states (lower m/z) than those formed from solutions in which the protein has signific...

Time of flight versus ion trap MS coupled to CE to analyse intact proteins

“…It is accepted that the shape of the charged distribution does mainly depend on the availability of the various chargeable basic sites (Arg, Lys, His and the N-terminus) [8], and thus to the tertiary structure of the protein (i. e. in a highly folded protein few basic sites will be available, however, in a fully unfolded state all basic sites should be accessible) [9]. This effect on the charge state distribution of protein has been verified studying the influence of acids [10], temperature [11] or solvents [12]. However, recently it has also been shown that proteins themselves can have an important influence on the response of other proteins [13].…”

Ionization Efficiency of Doubly Charged Ions Formed from Polyprotic Acids in Electrospray Negative Mode

“…Iavarone et al was the pioneer in studies of supercharging [12]. They observed an increase in the charge state for peptides if additives such as diethylamine, 2-methoxyethanol, ethylene glycol, glycerol, or m-nitrobenzyl alcohol were added to the solution [12][13][14].…”

Comparison of infrared multiphoton dissociation and collision-induced dissociation of supercharged peptides in ion traps

“…More highly charged peptides also have increased coulombic repulsion, which may facilitate dissociation compared with lower-charged species. Iavarone and Williams [35][36][37][38][39] explored the use of additives, such as m-nitrobenzyl alcohol (m-NBA), that increase the surface tension and reduce the vapor pressure of electrospray ionization (ESI) droplets, thus generating higher protein and peptide charge states. This phenomenon has been termed supercharging.…”