Joule Heating and Thermal Denaturation of Proteins in Nano-ESI Theta Tips
Electro-osmotically 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 electro-osmosis 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 electro-osmosis 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.
KeywordsJoule heating Theta tip Electro-osmosis Protein denaturation
Nano-electrospray ionization (nESI) is used to generate gaseous ions of biomolecules such as proteins, carbohydrates, lipids, etc. [1, 2]. Proteins normally result in multiply charged ions when subjected to electrospray ionization and charge state distributions are related to protein conformation. It is generally accepted, for example, the magnitude of charges is relatively high for unfolded conformations, which are described as high charge state distributions, while more folded conformations usually display relatively low charge state distributions [3, 4, 5]. For this reason, charge state distributions have been used in a biophysical context to monitor protein conformations using mass spectrometry. The magnitude of protein ion charge also has analytical implications. For example, high charge state ions are more efficiently detected by charge sensitive detectors like those used by Fourier transform based mass analyzers . Furthermore, increasing the charge state of a protein ion can lead to improved sequence coverage in top-down analysis [7, 8, 9], especially when electron transfer dissociation (ETD) and electron capture dissociation (ECD) are used as dissociation methods . Therefore, in-source protein denaturation can be desirable for the primary structural characterization of a protein via tandem mass spectrometry.
Tertiary and quaternary protein structures are stabilized by various interactions including salt bridges, hydrogen bonding, hydrophobic interactions and van der Waals interactions [11, 12]. These interactions can be affected by a variety of factors including temperature [4, 13, 14, 15], pH [16, 17, 18], ionic strength , solvent [20, 21, 22], surface effects , as well as instrumental parameters . Most methods intended to change protein conformation involve bulk solution manipulations, such as the addition of acid, base, organic solvent, supercharging reagents  or other additives, as well as heating. 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 [25, 26]. The electrothermal supercharging method manipulates protein conformation by changing the ionization voltage . 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.
Theta tips are nESI dual channel emitters that also function as micro-mixers prior to the ionization step . They are pulled from theta capillaries made of borosilicate glass that contain a septum in the center dividing a capillary into two separate channels into which different solutions can be loaded. A platinum wire is placed in each channel to apply spraying and mixing voltages. By applying the same ESI voltage to both channels, the solutions are sprayed out simultaneously and subsequently mix in the Taylor cone as well as in the ensuing droplets on a sub-millisecond time scale [29, 33]. This method has been applied to study protein unfolding and folding by mixing protein solutions with acid or ammonium acetate in the theta tip Taylor cone and droplets [29, 30, 32]. Due to the short mixing time, short-lived unfolding intermediates have been observed . A more recent study has shown that electro-osmotic flow can be induced between channels of a theta tip when applying differential voltages in the two channels . The duration and extent of mixing can be controlled by tuning the applied voltage and time of electro-osmosis. The solution phase mixing overcomes the reagent volatility limitation in the vapor exposure strategy [25, 27] and does not require a special mass spectrometric interface set up. However, the mixing step can alter the protein solution pH and composition if the two sides are mixed with dissimilar solutions. This is unfavorable for reagent or pH-sensitive studies like covalent modification and HDX . Here we demonstrate thermal denaturation of proteins in theta tips via Joule heating, which can be a useful way to manipulate protein conformation without altering solution composition.
Materials and Methods
Myoglobin from equine skeletal muscle, cytochrome c from bovine heart, cytochrome c from equine heart, ubiquitin from bovine erythrocytes, carbonic anhydrase II from bovine erythrocytes, and ammonium acetate were purchased from Sigma Aldrich (St. Louis, MO, USA). HPLC grade water was purchased from Fisher Scientific (Fair Lawn, NJ, USA). All proteins were dissolved in 0, 5, or 10 mM ammonium acetate solution at pH around 6 and the final protein concentration is 5–20 μM unless specifically noted. Proteins and chemical reagents were used without further purification.
Capillaries and Tip Holder
Dual channel borosilicate theta capillaries (1.5 mm o.d., 1.17 mm i.d., 0.165 mm septum thickness, 10 cm length) were purchased from Sutter Instrument Co. (Novato, CA, USA). Theta capillaries were pulled to theta tips (o.d. 10 μm) using a Flaming/Brown micropipette puller (P-87) from Sutter Instrument Co.
Solutions were loaded into both channels of a theta tip, which was held by a theta tip holder from Warner Instruments, LLC (Hamden, CT, USA), pictured in Supplemental Figure S-1. The original silver wires in the holder were replaced with Teflon coated platinum wires (A-M Systems, Sequim, WA, USA) to avoid discharge between the wires at the back of the theta capillary when voltages applied to the wires were different. The two wires were inserted into each channel of a theta tip to apply voltage to each side independently.
A quadrupole/time-of-flight (QqTOF) tandem mass spectrometer (QStar Pulsar XL; Sciex, Concord, ON, Canada) was used to perform all mass spectrometric experiments. The experimental procedure consists of four steps: electro-osmosis, ionization, dump spray, and mass analysis. In the electro-osmosis step the protein solution was electrically pumped back and forth between the two channels by grounding the wire in one theta tip channel while applying 100 ms of 10 Hz square wave voltage to the wire in the opposite channel. The square wave duty cycle is 50% and the voltage is ±100 V to ±500 V, where “±” was used to indicate the switch between positive and negative voltages during an electro-osmotic cycle. Next, the ionization step was triggered (1500 V on both wires, 80 ms), during which the ions are accumulated in Q2. The dump spray step was then triggered to spray out any residual analyte that had been exposed to the electro-osmosis step. For this purpose, a dump spray step of 200 ms was found sufficient to return the mass spectrum to that of the pre-osmosis step. The ion path voltages were set such that no ions were accumulated in Q2 during the dump spray step. Finally, the mass spectrum was recorded during the 150 ms mass analysis step. The power supplies and detailed trigger system are summarized in Supplemental Figure S-2.
Raman Temperature Measurements
Temperature measurements within the theta tip were conducted off-line using a set-up to simulate the theta tip arrangement in front of the mass spectrometer. To measure the solution temperature via Raman spectroscopy, the timing of the voltages applied to the wires in the theta tip was designed to simulate the various steps described above for the mass spectrometry experiments. The detailed triggering method is shown in Supplemental Figure S-3.
The temperature of the fluid near the apex of the theta tip was obtained noninvasively using Raman spectroscopy. The micro-Raman spectra were measured using a custom-built instrument that includes a 532 nm laser excitation (Coherent Sapphire SF CDRH 532 nm) and a TE-cooled CCD (Princeton Instruments SP2300). A 100× objective (Olympus LM Plan Fl) with a working distance of 3.4 mm was used to both focus the laser and collect the backscattered Raman signal. Laser power at the sample was set to 24.5 mW. Fine positional control was accomplished with a motorized microscope stage (Prior H101A/C). For the Raman experiments, both channels of the theta tip were filled with 5 mM ammonium acetate. Consecutive spectra with 100 ms exposure time were acquired while continuously cycling through the process of electro-osmosis-spray-simulated mass analysis. To obtain the training spectra for temperature calibration, a Pyrex 9530-3 borosilicate glass capillary (1.5–1.8 mm o.d., 90 mm length) was filled with 18.2 MΩ cm ultrapure water and heated using a Physitemp TS-4MPER thermal stage to temperatures between 20 °C and 90 °C, measured using a needle thermocouple (Physitemp MT-26/4 with an Omega DP701 reader). The temperature training spectra were collected with an integration time of 5 min per spectrum. Two representative training spectra corresponding to 30.3 °C and 74.3 °C are shown in Figure 5 insert (b).
The shape and intensity of the OH stretching mode of water is highly temperature-dependent and has been used in the past for Raman thermometry. For example, D’Arrigo et al. calibrated temperatures based on a ratio of OH stretch areas with respect to measured temperature values from a thermocouple . These areas were based on an approximate isosbestic point near 3400 cm–1, and the calibrated value was the ratio of the OH area to the left and to the right of that point. We employed an alternative hyperspectral procedure using self-modeling curve resolution (SMCR) to decompose the OH stretch into the two primary spectral components such that each spectrum is a linear combination of those two components. Each spectrum was first baseline-subtracted in the OH stretch region using a quadratic fit to user-defined points in the baseline on either side of the OH band. Next, using the baseline subtracted training spectra, a quadratic calibration curve relating measured temperature to a parameter representing the fractional spectral weight of the high temperature component in each measured spectrum was generated. Then for each experimental spectrum, a total least squares fit of the measured spectrum to the two SMCR components was used to quantify the fractional weight of the high temperature component, which was in turn converted to temperature using the training calibration curve. Two of these experimental spectra and their corresponding temperature values are shown in Figure 5 insert (b).
The Raman measurements were obtained asynchronously at a frame rate of about 6 fps, and subsequently synchronized with the applied voltage cycles to create plots similar to Figure 5 insert (a). Each curve of Figure 5 insert (a) includes 400 Raman measured temperature data points and approximately 110 cycles of electro-osmosis-spray-simulated mass analysis process.
Results and Discussion
Electro-osmosis Induced Protein Denaturation
Collectively, the results for these proteins showed that protein denaturation under conditions of electro-osmosis in a theta tip at relatively high square wave voltages is a general phenomenon. Furthermore, the extent of denaturation was found to be both condition-dependent (e.g., the magnitude of the square wave voltage) and protein-dependent (e.g., higher square wave voltages were required to denature proteins with higher melting temperatures). The results are consistent with thermal denaturation as a result of Joule heating at the end of the theta tip during electro-osmosis.
Influence of Ammonium Acetate Concentration on Protein Denaturation
Ammonium acetate is a commonly used additive in native mass spectrometry . High concentration of ammonium acetate displaces nonvolatile adducts and reduces the nonvolatile components influence . It also stabilizes the native conformation of proteins in solution through ionic specific interaction, which is normally referred to as Hofmeister effects [55, 56]. The protein–ligand dissociation constant also decreases with higher ammonium acetate concentration when the protein’s isoelectric point is higher than the pH of the solution . The isoelectric point of myoglobin is 6.8–7.4, whereas the 5 mM ammonium acetate solution pH is around 6. At this pH, ammonium acetate enhances the retention of the heme in the myoglobin binding pocket. Since holo-myoglobin stability is determined by the heme ligand affinity , the high binding affinity of the heme in ammonium acetate solution can further stabilize myoglobin.
Temperature Measurements Using Raman Spectroscopy
Figure 5 shows how the maximum solution temperature obtained during the electro-osmosis step is correlated to the applied square wave heating voltage. Based on the measured temperature, applying a ±200 V square wave to one channel increased the solution temperature to about 44 °C, whereas ±300 V voltage increased the solution temperature to 51 °C. Increasing the voltage amplitude to 500 V led to a maximum temperature at 77 °C. The small size of the theta tip and the associated large resistance imply that Joule heating is expected to produce a substantial temperature rise near the apex of a theta tip (the magnitude of which may be roughly estimated as described in the Supplemental Information), although heat dissipation exists in the open system. The Raman laser measuring point is about 8 μm away from the end of the tip and the true maximum temperature at the tip apex may also be underestimated. Nevertheless, the Raman measurements directly show that the electro-osmosis process gives rise to an increase in the solvent temperature that is directly related to the square wave voltage applied to induce electro-osmosis.
Correlation Between Protein Melting Temperature and Denaturation Voltage
In this report, we demonstrate protein denaturation resulting from electro-osmosis in a theta tip nano-ESI capillary. The effect is shown to arise from Joule heating via both direct and indirect evidence. Indirect evidence included an increase in the extent of protein denaturation with the magnitude of the voltage of a square wave used to effect electro-osmosis. This effect was demonstrated for myoglobin, equine cytochrome c, and carbonic anhydrase II solutions. Joule heating is expected to increase with field strength. An increase in the extent of denaturation for myoglobin was also observed with an increase in the ammonium acetate concentration. Joule heating is expected to increase with solution conductivity. Using Raman spectroscopy temperature measurements near to the capillary tip, an increase in solution temperature, direct evidence for Joule heating, was found to correlate with the amplitude of the square wave voltage. Electro-osmosis-induced Joule heating was observed to be positively correlated to protein melting temperature when a solution of a mixture of proteins of known melting temperature was subjected to a series of experiments with increasing square wave heating voltage. This work points to the development of a convenient and efficient way to modulate solution temperature in a nano-ESI theta tip prior to spraying into a mass spectrometer. It represents a flexible approach for controlled protein denaturation that does not depend on changes in solution additives or solvent composition. With further development, this effect may serve as the basis for a method to study protein thermal stabilities on small quantities of materials and with mixtures of proteins. Given the ability to alter temperatures in a pulsed fashion on the time-scales of tenths of seconds, this effect may also prove to be useful in studying protein unfolding and refolding dynamics on such a time-scale.
This work was supported by the National Institutes of Health under grant GM R37-45372. Support for F.Z. was provided by a W. Brooks Fortune Fellowship in Analytical Chemistry.
- 8.Jockusch, R.A., Schnier, P.D., Price, W.D., Strittmatter, E.F., Demirev, P.A., Williams, E.R.: Effects of charge state on fragmentation pathways, dynamics, and activation energies of ubiquitin ions measured by Blackbody infrared radiative dissociation. Anal. Chem. 69, 1119–1126 (1997)CrossRefGoogle Scholar
- 19.Ohyashiki, T., Taka, M., Mohri, T.: The effects of ionic strength on the protein conformation and the fluidity of porcine intestinal brush border membranes. J. Biol. Chem. 260, 6857–6861 (1985)Google Scholar
- 27.Girod, M., Antoine, R., Dugourd, P., Love, C., Mordehai, A., Stafford, G.: Basic vapor exposure for tuning the charge state distribution of proteins in negative electrospray ionization: elucidation of mechanisms by fluorescence spectroscopy. J. Am. Soc. Mass Spectrom. 23, 1221–1231 (2012)CrossRefGoogle Scholar
- 37.Upadhyay, S.K.: Chemical Kinetics and Reaction Dynamics. Springer: New York City, NY; Anamaya Publishers: New Delhi, India (2006)Google Scholar
- 38.Grossman, P.D., Colburn, J.C.: Capillary Electrophoresis: Theory and Practice. Academic Press, Inc., Cambridge, MA (1992)Google Scholar
- 39.Whatley, H.: Basic principles and modes of capillary electrophoresis. In: Petersen, J.R., Mohammad, A.A. (eds.) Clinical and Forensic Applications of Capillary Electrophoresis, p. 37. Humana Press Inc, Totowa, NJ (2001)Google Scholar
- 41.Sarraf, N., Saboury, A., Ranjbar, B., Moosavi-Movahedi, A.: Structural and functional changes of bovine carbonic anhydrase as a consequence of temperature. Acta Biochim. Pol. 51, 665–671 (2004)Google Scholar
- 45.Saito, R., Sato, T., Ikai, A., Tanaka, N.: Structure of bovine carbonic anhydrase II at 1.95 A resolution. Acta Crystallogr. D60, 792–795 (2004)Google Scholar
- 52.Bágel’ová, J., Antalík, M., Tomori, Z.: Effect of polyglutamate on the thermal stability of ferricytochrome c. Biochem. Mol. Biol. Int. 43, 891–900 (1997)Google Scholar