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

Abstract

The exposure of electrospray droplets generated from either highly acidic or highly basic solutions to basic or acidic vapors, respectively, admitted into the counter-current drying gas, has been shown to lead to significant changes in the observed charge state distributions of proteins. In both cases, distributions of charge states changed from relatively high charge states, indicative of largely denatured proteins, to lower charge state distributions that are more consistent with native protein conformations. Ubiquitin, cytochrome c, myoglobin, and carbonic anhydrase were used as model systems. In some cases, bimodal distributions were observed that are not noted under any solution pH conditions. The extent to which changes in charge state distributions occur depends upon the initial solution pH and the pK a or pK b of the acidic or basic reagent, respectively. The evolution of charged droplets in the sampling region of the mass spectrometer inlet aperture, where the vapor exposure takes place, occurs within roughly 1 ms. The observed changes in the spectra, therefore, are a function of the magnitude of the pH change as well as the rates at which the proteins can respond to this change. The exposure of electrospray droplets in this fashion may provide means for accessing transient folding states for further characterization by mass spectrometry.

Ion Mobility-Mass Spectrometry Reveals Highly-Compact Intermediates in the Collision Induced Dissociation of Charge-Reduced Protein Complexes

“…Methods for reducing analyte ion charge states range from simple solution manipulation approaches to more complex gasphase ion-neutral and ion-ion reactions [33][34][35][36][37][38][39][40][41], with advantages and disadvantages for each. Solution-phase manipulation approaches for charge reduction have the advantages of simplicity and efficiency; however, such protocols also often sacrifice native protein-protein interactions, as well as the overall effectiveness of the charge reduction process [33][34][35].…”

“…Alternatively, gas-phase charge reduction approaches do not affect protein-protein interactions; however, require specialized instrument modifications and typically sacrifice molecular efficiency, in that charge reduction agents must often be added in large excess to significantly reduce ion charge [35][36][37][38][39][40][41].…”

Basic Vapor Exposure for Tuning the Charge State Distribution of Proteins in Negative Electrospray Ionization: Elucidation of Mechanisms by Fluorescence Spectroscopy

“…In addition, Schnier and coworkers [31] and, recently, McLuckey and coworkers [32][33][34] reported a simple vapor introduction technique in the atmosphere/vacuum interface region of an ESI source. Using this setup, the average charge state of ions derived from a protein was increased in the positive polarity via the introduction of acidic vapors and increased in the negative polarity via the introduction of basic vapors.…”

Joule Heating and Thermal Denaturation of Proteins in Nano-ESI Theta Tips

“…These methods can be time consuming and require larger sample volumes. In recent years, fast conformation manipulation methods in conjunction with ESI have been developed, including vapor exposure [25, 26, 27], electrothermal denaturation[24, 28], and theta tip mixing. [29, 30, 31, 32] During vapor exposure, the ESI droplets containing the protein are allowed to interact with acidic or basic vapors added to the nitrogen curtain gas, leading to protein denaturation or refolding on the basis of pH changes.…”

“…[29, 30, 31, 32] During vapor exposure, the ESI droplets containing the protein are allowed to interact with acidic or basic vapors added to the nitrogen curtain gas, leading to protein denaturation or refolding on the basis of pH changes. [25, 26] The electrothermal supercharging method manipulates protein conformation by changing the ionization voltage. [24] It has been reasoned that by applying a high spray voltage the droplet size is increased, thereby elongating its lifetime in the hot capillary interface and maximizing the thermal denaturation of the protein in the droplet.…”