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

Abstract

Electroosmotically-induced Joule heating in theta tips and its effect on protein denaturation were investigated. Myoglobin, equine cytochrome c, bovine cytochrome c and carbonic anhydrase II solutions were subjected to electroosmosis in a theta tip and all of the proteins were denatured during the process. The extent of protein denaturation was found to increase with the applied square wave voltage and electrolyte concentration. The solution temperature at the end of a theta tip was measured directly by Raman spectroscopy and shown to increase with the square wave voltage, thereby demonstrating the effect of Joule heating through an independent method. The electroosmosis of a solution comprised of myoglobin, bovine cytochrome c, and ubiquitin demonstrated that the magnitude of Joule heating that causes protein denaturation is positively correlated with protein melting temperature. This allows for a quick determination of a protein’s relative thermal stability. This work establishes a fast, novel method for protein conformation manipulation prior to MS analysis and provides a temperature-controllable platform for the study of processes that take place in solution with direct coupling to mass spectrometry.

Direct Mass Spectrometry Analysis of Complex Mixtures by Nanoelectrospray with Simultaneous Atmospheric Pressure Chemical Ionization and Electrophoretic Separation Capabilities

“…Figure 1b compares tip stability under different spray conditions. Joule heating 24 (among other factors) generated after applying 5−8 kV to an electrode in contact with analyte solution (conventional nESI) is mainly responsible for the tip burning/breakage observed here. For example, the electric field at the emitter apex is a key parameter in controlling electrospray current, which in turn determines the intensity of heat generated.…”

Large-Area Triboelectric Nanogenerator Mass Spectrometry: Expanded Coverage, Double-Bond Pinpointing, and Supercharging

“…Electrothermal supercharging represents an alternative means to induce protein ACS increases in native mass spectrometry and is a function of the voltage applied during the electrospray process, among other factors . This phenomenon is the result of protein unfolding following thermal denaturation induced by high electric fields . While results shown in Figure a were obtained under denaturing conditions, native MS conditions were also tested with TENG MS.…”

“…43 This phenomenon is the result of protein unfolding following thermal denaturation induced by high electric fields. 44 While results shown in Figure 6a were obtained under denaturing conditions, native MS conditions were also tested with TENG MS. Under these conditions, both Cyt C and myoglobin showed a shift toward higher charge states that increased at shorter emitter-inlet distances, making unfolded ion structures more abundant (Figure 7).…”

Thermal Analysis of a Mixture of Ribosomal Proteins by vT-ESI-MS: Toward a Parallel Approach for Characterizing the Stabilitome

“…Recently, several groups have developed and applied vT-ESI-MS methods to study the temperature dependence of protein (un)­folding, − protein–ligand interactions in solution, , as well as kinetic studies that have led to a detailed understanding of the driving forces associated with structural transitions . These studies suggest that it should be possible to analyze mixtures of intact proteins and protein complexes in parallel by following changes in charge state distributions with temperature. , While the relatively well-defined nature of the ribosomal protein mixture described in this study allows us to characterize different species based on precursor mass characterization, we note that the approach is well suited for top-down MS identification. − The proof-of-concept study presented below indicates that structural transitions of native protein monomers, as well as protein–ligand and protein–protein complexes, can be determined.…”