Melting proteins confined in nanodroplets with 10.6 μm light provides clues about early steps of denaturation

Abstract

Ubiquitin confined within nanodroplets was irradiated with a variable-power CO2 laser. Mass spectrometry analysis shows evidence for a protein "melting"-like transition within droplets prior to solvent evaporation and ion formation. Ion mobility spectrometry reveals that structures associated with early steps of denaturation are trapped because of short droplet lifetimes.

Figure 1

Schematic diagram of the instrument. Droplets diameters are estimated to be ~0.05 and 1.0 μm when produced from small ~1.0 and 20 μm dia. ESI capillary emitters (see Experimental section in the Supplementary Information). For 10 μM ubiquitin solutions, we estimate that only one in three of the droplets contains a protein molecule. After their formation by electrospray, droplets pass through a CO₂ laser beam focused at the immediate entrance to the instrument orifice. Activation in this region may induce structural changes in the protein which leads to changes in the protein charge state distribution and ion structures. This instrument is also equipped with a ZnSe window in the middle of the drift tube, which allows ions of a known mobility to be excited with 10.6 μm radiation. A series of control experiments in which the laser is focused through the drift tube shows that gaseous protein ions are not activated in the absence of solvent at the laser powers used (See Supplementary Information). This approach has similarities with an elegant “laser spray” technique, in which 10.6 μm light from an IR laser was focused into the metal-capillary tip of an ESI source to heat the bulk solution inside the capillary (see ref. for details).

Figure 2

(top) Average charge state as a function of solution temperature (squares) (from ref. 9) and laser power (open circles) for ubiquitin in aqueous solution at pH 3. Solid lines show the best fit of the data assuming a two-state model with T_m = 71 ± 2 °C and T_p =10.4 ± 0.3 W. Insets show representative mass spectra at different solution temperatures and laser powers. Upon blocking the radiation, a melted charge state distribution immediately returns to the room temperature distribution, indicating that laser excitation heats only the droplets. The bottom plot shows ubiquitin ions heated in droplets at pH 2.5 (upside down triangles), pH 3.0 (open circles), and pH 4.0 (diamonds) with melting transitions of T_p = 5.8 ± 0.3, 10.4 ± 0.3, and 11.8 ± 0.3 W, respectively.

Figure 3

Cross section distributions for (a) [M+7H]⁷⁺, (b) [M+8H]⁸⁺, and (c) [M+11H]¹¹⁺ ions of ubiquitin at different temperatures (purple) and laser powers, with black and red lines corresponding to structures that form upon irradiating droplets formed from ~1 μm and ~20 μm electrospray emitters, respectively. Structures in (c) are adapted from reference .