Conformations and folding of lysozyme ions in vacuo.

Identifying the key factor(s) governing the overall protein charge is crucial for the interpretation of electrospray-ionization mass spectrometry data. Current hypotheses invoke different principles for folded and unfolded proteins. Here, first we investigate the gas-phase structure and energetics of several proteins of variable size and different folds. The conformer and protomer space of these proteins ions is explored exhaustively by hybrid Monte-Carlo/molecular dynamics calculations, allowing for zwitterionic states. From these calculations, the apparent gas-phase basicity of desolvated protein ions turns out to be the unifying trait dictating protein ionization by electrospray. Next, we develop a simple, general, adjustable-parameter-free model for the potential energy function of proteins. The model is capable to predict with remarkable accuracy the experimental charge of folded proteins and its well-known correlation with the square root of protein mass.

Section: Gas-phase Basicity and Protein Ionizationmentioning

confidence: 72%

A Computational Model for Protein Ionization by Electrospray Based on Gas-Phase Basicity

Marchese

Grandori

Carloni

et al. 2012

Section: Solvent Evaporation Versus Gas-phase Collisionsmentioning

confidence: 99%

“…We are not able to differentiate between these mechanisms from these experiments alone. However, these two processes can be independently studied by examining either the gas-phase ion-molecule chemistry of isolated protein ions [24,[26][27][28] or by storing solvated ions at ultrahigh vacuum using trapping instruments, such as the Fourier-transform mass spectrometer, to directly observe the solvent evaporation process [29,30].In the solvent evaporation process, the most volatile solvent preferentially leaves, resulting in a solvated analyte ion in which the solvent is enhanced in the least volatile component. Ridge and co-workers [17] proposed that the maximum charge state of an ion should then be determined by the GB of the least volatile solvent.…”

mentioning

confidence: 99%

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

Iavarone

Jurchen

Williams

2000

172

199

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...

“…In contrast, mass spectrometry requires little sample, and with electrospray ionization (ESI) or soft laser desorption/ionization (John Fenn and Kiochi Tanaka 2002 in chemistry), a wide variety of biomolecules and specific complexes can be introduced into a mass spectrometer. A number of methods have been developed to probe the general three-dimensional shapes of biomolecule ions and noncovalent complexes using the techniques of ion mobility [1][2][3][4][5][6][7][8], H/D exchange both in solution [9,10] and the gas phase [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28], and proton-transfer reactivity [29,30].…”

mentioning

confidence: 99%

Further studies on the origins of asymmetric charge partitioning in protein homodimers

Jurchen

García

Williams

2004

115

Dissociation of gas-phase protonated protein dimers into their constituent monomers can result in either symmetric or asymmetric charge partitioning. Dissociation of ␣-lactalbumin homodimers with 15ϩ charges results in a symmetric, but broad, distribution of protein monomers with charge states centered around 8ϩ/7ϩ. In contrast, dissociation of the 15ϩ heterodimer consisting of one molecule in the oxidized form and one in the reduced form results in highly asymmetric charge partitioning in which the reduced species carries away predominantly 11ϩ charges, and the oxidized molecule carries away 4ϩ charges. This result cannot be adequately explained by differential charging occurring either in solution or in the electrospray process, but appears to be best explained by the reduced species unfolding upon activation in the gas phase with subsequent separation and proton transfer to the unfolding species in the dissociation complex to minimize Coulomb repulsion. For dimers of cytochrome c formed directly from solution, the 17ϩ charge state undergoes symmetric charge partitioning whereas dissociation of the 13ϩ is asymmetric. Reduction of the charge state of dimers with 17ϩ charges to 13ϩ via gas-phase proton transfer and subsequent dissociation of the mass selected 13ϩ ions results in a symmetric charge partitioning. This result clearly shows that the structure of the dimer ions with 13ϩ charges depends on the method of ion formation and that the structural difference is responsible for the symmetric versus asymmetric charge partitioning observed. This indicates that the asymmetry observed when these ions are formed directly from solution must come about due either to differences in the monomer conformations in the dimer that exist in solution or that occur during the electrospray ionization process. These results provide additional evidence for the origin of charge asymmetry that occurs in the dissociation of multiply charged protein complexes and indicate that some solution-phase information can be obtained from these gas-phase dissociation experiments. (J Am Soc Mass