A versatile microfluidic chip for millisecond time-scale kinetic studies by electrospray mass spectrometry

Rob, Tamanna; Wilson, Derek J.

doi:10.1016/j.jasms.2008.09.005

Cited by 52 publications

(79 citation statements)

References 39 publications

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“…The kinetics of acid-induced cytochrome c unfolding from one charge state to the next observed in the present investigation qualitatively agrees with previously reported studies of the unfolding kinetics of this protein (10). It is interesting that the time constant increased as the charge state increased during the unfolding event.…”

Section: Discussionsupporting

confidence: 92%

“…Acid-induced unfolding of cytochrome c was chosen for a proof-of-principle experiment because this process has been previously well characterized (10,(22)(23)(24)(25)(26)(27) at different pH values (Fig. S4).…”

Section: Resultsmentioning

confidence: 99%

“…Another approach is to stimulate the rapid initiation of a reaction by photo-triggered initiation (4), electron transfer (5), or temperature jump/rise (6). A small-size reactor was also used for rapid mixing so that the time required for diffusion-dependent mixing is minimized (7)(8)(9)(10).…”

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confidence: 99%

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Microdroplet fusion mass spectrometry for fast reaction kinetics

Lee

Kim

Nam

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

210

258

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We investigated the fusion of high-speed liquid droplets as a way to record the kinetics of liquid-phase chemical reactions on the order of microseconds. Two streams of micrometer-size droplets collide with one another. The droplets that fused (13 μm in diameter) at the intersection of the two streams entered the heated capillary inlet of a mass spectrometer. The mass spectrum was recorded as a function of the distance x between the mass spectrometer inlet and the droplet fusion center. Fused droplet trajectories were imaged with a high-speed camera, revealing that the droplet fusion occurred approximately within a 500-μm radius from the droplet fusion center and both the size and the speed of the fused droplets remained relatively constant as they traveled from the droplet fusion center to the mass spectrometer inlet. Evidence is presented that the reaction effectively stops upon entering the heated inlet of the mass spectrometer. Thus, the reaction time was proportional to x and could be measured and manipulated by controlling the distance x. Kinetic studies were carried out in fused water droplets for acid-induced unfolding of cytochrome c and hydrogen-deuterium exchange in bradykinin. The kinetics of the former revealed the slowing of the unfolding rates at the early stage of the reaction within 50 μs. The hydrogendeuterium exchange revealed the existence of two distinct populations with fast and slow exchange rates. These studies demonstrated the power of this technique to detect reaction intermediates in fused liquid droplets with microsecond temporal resolution.mass spectrometry | liquid microdroplets | reaction kinetics | protein unfolding | hydrogen-deuterium isotope exchange T ime-resolved measurements of reaction intermediates are crucial for understanding the fast kinetics of chemical reactions. Various approaches have been implemented to improve the temporal resolution of kinetic measurements in liquid reactions (1, 2), which are often limited by the mixing time. One approach for improving the mixing time involves the use of turbulent flow to increase the shear stress in fluid channels (3). Another approach is to stimulate the rapid initiation of a reaction by photo-triggered initiation (4), electron transfer (5), or temperature jump/rise (6). A small-size reactor was also used for rapid mixing so that the time required for diffusion-dependent mixing is minimized (7-10).Among various methods for detecting reaction intermediates, mass spectrometry has been a powerful tool for probing reaction products because it can discriminate similar species by their mass-to-charge ratio while simultaneously measuring multiple species. Time-resolved mass spectrometry (11) has been widely used for measuring the kinetics of protein-ligand complexation, organometallic compound formation, and enzyme-catalyzed processes. Despite these efforts for improving temporal resolution, time-resolved mass spectrometry has been limited to the millisecond timescale, with a recent achievement of 300 μs (12).A major obstacle for imp...

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Section: Discussionsupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

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Microdroplet fusion mass spectrometry for fast reaction kinetics

Lee

Kim

Nam

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

210

258

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“…Microfluidic Device Fabrication-The microfluidic device was constructed as described previously (32,33). Briefly, the microfluidic channel design was generated in CorelDraw X3 (Corel Co., Ottawa, Ontario, Canada) and lasered onto blank precut polymethyl methacrylate (PMMA) 4 substrate (8.9 ϫ 3.8 ϫ 0.6 cm; Professional Plastics, Fullerton, CA).…”

Section: Methodsmentioning

confidence: 99%

Conformational Dynamics and Allostery in Pyruvate Kinase

Donovan

Zhu

Liuni

et al. 2016

Journal of Biological Chemistry

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Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the "domain rotation model" suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the "rigid body reorientation model" proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogendeuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1,6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1,6-bisphosphate destabilizes an ␣-helix that bridges the allosteric and active site domains within the monomeric unit. This destabilizes the ␤-strands within the (␤/␣) 8 -barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1,6-bisphosphate affects the active site.

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“…Microfluidic devices for studying kinetics have been implemented on both capillary- 19 and chipbased platforms. 20 The second functional unit is the rapid mixing apparatus, which can be static 21 or adjustable, 19,22 turbulent or diffusive. 23 Finally, there is the detector that can be either integrated into the device (i.e., "on-chip" analysis) or separate but coupled to the device (i.e., "off-chip" analysis).…”

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confidence: 99%