Laser Heating Nanoelectrospray Emitters for Fast Protein Melting Measurements with Mass Spectrometry

Abstract

Temperature-controlled nanoelectrospray ionization has been used to measure heat-induced conformational changes of biomolecules by mass spectrometry, but long thermal equilibration times associated with heating or cooling an entire emitter limit how fast these data can be acquired. Here, the tip of a borosilicate electrospray emitter is heated using 10.6 μm light from an unfocused CO2 laser. At 1.2 W, the solution inside the emitter tip can be heated from room temperature to a steady-state temperature of 78.2 ± 2.5 °C in less than 0.5 s and cools from 82.6 ± 0.6 °C back to room temperature within 4 s. The time required to establish a steady-state temperature is more than 100-fold faster than that required for a resistively heated emitter due to the low thermal mass. Protein unfolding curves measured as a function of laser power can be acquired in ∼40 s compared to a resistively heated apparatus that required ∼21 min to acquire similar data. Laser power is calibrated to temperature by comparisons of the average charge state of the protein cytochrome c measured with laser heating and with resistive heating. This laser heating method is applied to a three-component protein mixture to demonstrate the ability to rapidly acquire melting temperatures of proteins in mixtures. The ability to rapidly assess the thermal stabilities of multiple proteins simultaneously shows significant promise for coupling temperature-controlled electrospray ionization (ESI) to separation techniques, providing a high-throughput method for determining the effects of solution composition, drug binding, or sequence mutations on protein thermal stability.

Overcoming aggregation with laser heated nanoelectrospray mass spectrometry: thermal stability and pathways for loss of bicarbonate from carbonic anhydrase II

“…The laser power was ramped between 0 W (laser off ) and ∼3.9 W. The laser powers used here are higher than those used in prior LH-ESI experiments due to irradiating a shorter, thinner volume at the end of the emitter tip. 58 The mass spectra did not change with laser powers below ∼1.8 W. At ∼1.8 W, higher charge state ions (+11-+31) corresponding to denatured BCAII appear. These high charge state ions increase in abundance with increasing laser power (Fig.…”

“…The +6-+8 charge states of cyt c are also formed, consistent with prior results. 35,58,69 The melting of BCAII and cyt c was measured between solution temperatures of 27 °C and 86 °C using vT-ESI. At ∼64 °C, there is a small population of higher charge states corresponding to unfolded BCAII (Fig.…”

“…39 Rapid heating of a solution on timescales between 0.8 ms and 4 minutes can also be done using theta glass emitters 55 or by rapidly flowing a solution through a resistively heated portion of an emitter. 35,47 Protein melting has also been investigated using laser heating, either by irradiating the end of electrospray emitters [56][57][58] or by heating electrospray droplets directly. 38,59 Heating the end of a 100 μm diameter metal emitter led to protein unfolding and dissociation of noncovalent biomolecular complexes.…”

“…56,57,60 Heating the end of borosilicate emitters has the advantage that the solution temperature can be rapidly changed due to the small solution volume and rapid heat transfer to the surrounding atmosphere. 58 Solutions can be heated from room temperature to ∼78 °C in less than 0.5 s and cooled from ∼82 °C back to room temperature in <4 s. 58 The rapid equilibration enables acquisition of protein melting curves in <40 s and laser power can be converted to solution temperature by calibration with vT-ESI data. 58 By laser heating electrospray droplets, unfolding intermediates were kinetically trapped and analyzed by ion mobility spectrometry.…”

“…58 Solutions can be heated from room temperature to ∼78 °C in less than 0.5 s and cooled from ∼82 °C back to room temperature in <4 s. 58 The rapid equilibration enables acquisition of protein melting curves in <40 s and laser power can be converted to solution temperature by calibration with vT-ESI data. 58 By laser heating electrospray droplets, unfolding intermediates were kinetically trapped and analyzed by ion mobility spectrometry. 38,59 Multiple time points along the unfolding process were measured using electrospray emitters with different diameters to produce droplets with different lifetimes.…”