Ultrafast (1 μs) Mixing and Fast Protein Folding in Nanodrops Monitored by Mass Spectrometry

Abstract

The use of theta-glass emitters and mass spectrometry to monitor reactions that occur as fast as one μs is demonstrated. Acidified aqueous solutions containing unfolded proteins are mixed with aqueous ammonium acetate solutions to increase the solution pH and induce protein folding during nanoelectrospray ionization. Protein charge-state distributions show the extent to which folding occurs, and reaction times are obtained from known protein folding time constants. Shorter reaction times are obtained by decreasing the solution flow rate, and reaction times between 1.0 and 22 μs are obtained using flow rates between 48 and 2880 pL/s, respectively. Remarkably similar reaction times are obtained for three different proteins (Trp-cage, myoglobin, and cytochrome c) with folding time constants that differ by more than an order of magnitude (4.1, 7, and 57 μs, respectively), indicating that the reaction times obtained using rapid mixing from theta-glass emitters are independent of protein identity. A folding time constant of 2.2 μs is obtained for the formation of a β-hairpin structure of renin substrate tetradecapeptide, which is the fastest folding event measured using a rapid mixing technique. The 1.0 μs reaction time obtained here is about an order of magnitude lower than the shortest reaction time probed using a conventional mixer (8 μs). Moreover, this fast reaction time is obtained with a 48 pL/s flow rate, which is 2000-times less than the flow rate required to obtained the 8 μs reaction time using a conventional mixer. These results indicate that rapid mixing with theta-glass emitters can be used to access significantly faster reaction times while consuming substantially less sample than in conventional mixing apparatus.

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

“…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.…”

“…[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. [29] A more recent study has shown that electroosmotic flow can be induced between channels of a theta tip when applying differential voltages in the two channels.…”

Microsecond and nanosecond polyproline II helix formation in aqueous nanodrops measured by mass spectrometry

“…26,27 A 2.2 μs folding time constant for the formation of a β-hairpin in a 14 residue peptide was determined, which is the fastest folding event that has been directly measured with a rapid mixing technique. 27 Reaction times of between 1 and 22 μs have been achieved with thes e devices by varying the solution flow rate, which depends on the backing pressure and on the nanoESI-emitter tip size. 27 Here, rapid mixing with theta-glass emitters is used to investigate the rates of formation for PPII helices in two short (16 and 21 re sidue) peptides.…”

“…27 Reaction times of between 1 and 22 μs have been achieved with thes e devices by varying the solution flow rate, which depends on the backing pressure and on the nanoESI-emitter tip size. 27 Here, rapid mixing with theta-glass emitters is used to investigate the rates of formation for PPII helices in two short (16 and 21 re sidue) peptides. These peptides are prepared in acidified aqueous solutions in which they are highly unfolded and mixed with buffered aqueous solutions during nanoESI to increase the solution pH and induce the formation of the PPII helices.…”

“…Grounded platinum wires are brought into contact with the solutions in the emitters, and a backing pressure of either 5 or 10 psi is applied to the back end of the capillary, depending on the desired reaction time. 27 NanoESI is initiated by applying about a −700 V potential to the heated capillary of the ESI interface. Average charges are computed as abundance weighted sums of the charge states, and uncertainties are standard deviations from triplicate measurements.…”