Investigation of deprotonation reactions on globular and denatured proteins at atmospheric pressure by ESSI-MS

Abstract

Deprotonation reactions of multiply charged protein ions have been studied by introducing volatile reference bases at atmospheric pressure between an electrosonic spray ionization (ESSI) source and the inlet of a mass spectrometer. Apparent gas-phase basicities (GB app ) of different charge states of protein ions were determined by a bracketing approach. The results pp obtained depend on the conformation of the protein ions in the gas phase, which is linked to the type of buffer used (denaturing or nondenaturing [1] rapidly became established as the method of choice for the production of large biomolecular ions in the gas phase. For proteins, ESI leads to the formation of multiply charged ions, both in the positive and negative ion modes. The charge-state distribution is directly related to the conformation of the macromolecular ions [2] and the proton transfer reactions in the gas phase [3]. Thermodynamic information, such as values for the gas-phase acidity/basicity (GA/GB), is essential to the understanding of the proton transfer processes and generation of ions by ESI in general. The intrinsic gas-phase basicity of a [M ϩ (n Ϫ 1)H] (n Ϫ 1)ϩ ion or the gas-phase acidity of a [M ϩ nH] nϩ ion can be defined as the free enthalpy change ⌬G 0 of the reaction:Since this reaction proceeds with a reverse activation barrier, due to Coulombic repulsion between the two charges product ions, the terms "apparent gas-phase acidity" (GA app ) and "apparent gas-phase basicity" (GB app ) are more appropriate. The GA app is defined as the sum of the ⌬G and the energy of the activation barrier, considered as a negative term [4].Determination of GB app s of peptides and proteins provides important information which is analogous to measurements of pKa in solution. For example, pKa values are greatly affected by intramolecular interactions, and the conversion of the denatured to the native form maximizes these interactions. Similarly, GB app values will also reflect the conformation of the ions in the gas phase.For more than 10 years, research groups have tried to determine apparent gas-phase basicities of peptides and proteins. For small peptides, like bradykinin and its des-arginine analogues, the kinetic method can be directly used to determine GB app values [5,6]. The energy of the activation barrier was obtained by measuring the kinetic energy release to calculate the intrinsic GB of two different charge states. Unfortunately, this method is quite difficult to transfer to multiply charged protein ions.A bracketing technique, consisting of evaluating the qualitative or quantitative rate of proton transfer between a neutral volatile base and protein ions was introduced to measure GB app values of multiply charges ions [4,7,8]. In terms of instrumentation, different systems can be used to observe the deprotonation reactions. High-pressure mass spectrometry (HPMS) experiments were designed by the group of Kebarle to study cluster formation from small organic or inorganic ions, and water in particular [9,10]. This apparatus was...

Effects of ion/ion proton transfer reactions on conformation of gas-phase cytochrome c ions

“…The ion conformations formed via charge reduction reactions at relatively low-pressure inside the ion trap could also be compared with those formed from ion/ molecule reactions with neutral base gases in the ESI ion source, using reactions described by Zenobi [54,55]. This experiment could probe the time frame over which ions liberated by ESI assume their final conformations, which is important to the key issue of whether the conformations of the ions produced by ESI resemble those in the solution [10,31,32,47].…”

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

“…The extent of protein ionization has been interpreted in terms of GB also in this case [6][7][8][9]. In particular, the GB app of folded cytochrome c has been calculated from the crystallographic structure, accounting for Coulomb repulsions [9].…”

Vapor Treatment of Electrospray Droplets: Evidence for the Folding of Initially Denatured Proteins on the Sub-Millisecond Time-Scale

“…Also, proton transfer in the gas phase after the proteins have left the droplet could play a role in the observed charge state distributions. However, the presence of distinct bimodal distributions (see Figure 2c and Figure 6b) rather than a gradual stepwise change from one charge state distribution to another, often seen with gas-phase proton transfer reactions [32][33][34][35][36][37][38][39][40][41][42], suggests that the observed changes in the protein charge state distributions cannot be fully rationalized on the basis of gas-phase reactions. However, contributions from gas-phase proton transfer, at least to some extent, cannot be ruled out.…”

“…In addition, extractive electrospray ionization (EESI) has employed basic solvents infused at rates of 0.1-2 μL/min resulting in ion/molecule reactions in open air in the region between the basic reagent spray and the electrospray of the protein or peptide analyte [39]. Zenobi et al have also published work using electrosonic spray ionization (ESSI) [40] in which volatile gaseous vapors were introduced to reduce the observed charge state. However, they were unsuccessful when conducting similar experiments with ESI or nano-ESI [41].…”