Stopped-flow electrospray ionization mass spectrometry: a new method for studying chemical reaction kinetics in solution

Kolakowski, Beata; Simmons, Douglas A.; Konermann, Lars

doi:10.1002/(sici)1097-0231(20000515)14:9<772::aid-rcm942>3.0.co;2-k

Cited by 43 publications

(13 citation statements)

References 23 publications

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“…To test this hypothesis, we measured the response to 0.3 lM ACh at )50 mV before and after a 12-h incubation in a nearly saturating ACh concentration (300 lM, see Figl et al 1998) followed by a 24-h incubation in 2 mL of ACh-free media (Figs 5a-d). ACh spontaneously hydrolyzes in buffered saline but ACh hydrolysis at the pH, temperature, and ionic strength of our media is far too slow to significantly reduce the ACh concentration of our incubation media over a 12-h period (Kolakowski et al 2000). Xenopus oocytes also have endogenous acetylcholinesterase activity but the acetylcholinesterase activity of a Stage V-VI oocyte would reduce the ACh concentration of 2 mL of 300 lM ACh by < 0.01% during a 12-h incubation (Gundersen and Miledi 1983).…”

Section: Xenopus Oocytes Accumulate and Release [ 3 H]nicotinementioning

confidence: 97%

“…1998) followed by a 24‐h incubation in 2 mL of ACh‐free media (Figs 5a–d). ACh spontaneously hydrolyzes in buffered saline but ACh hydrolysis at the pH, temperature, and ionic strength of our media is far too slow to significantly reduce the ACh concentration of our incubation media over a 12‐h period (Kolakowski et al . 2000).…”

Section: A 12‐h Incubation In 300 µM Ach Does Not Persistently Depresmentioning

confidence: 99%

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Nicotine trapping causes the persistent desensitization of α4β2 nicotinic receptors expressed in oocytes

Flotildes

et al. 2003

Journal of Neurochemistry

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To determine whether prolonged nicotine exposure persistently inactivates rat a4b2 nicotinic receptors expressed in Xenopus oocytes, we measured the voltage-clamped a4b2 response to acetylcholine (ACh) before and 24 h after, 1-h or 12-h incubations in 10 lM nicotine. A 12-h incubation in 10 lM nicotine depressed the a4b2 ACh response for 24 h without affecting total or surface a4b2 expression. To determine whether oocyte-mediated nicotine release caused this depression, we co-incubated an a4b2-expressing oocyte with an un-injected one (pre-incubated in 10 lM nicotine for 12 h) for 24 h and measured the change in the a4b2 ACh response. The response decreased by the same factor after the co-incubation as it did after a 12-h incubation in 10 lM nicotine and a 24-h incubation in nicotine-free media. Thus, oocytemediated nicotine release caused the persistent desensitization we observed after a 12-h incubation in 10 lM nicotine. Prolonged nicotine exposure persistently depresses the agonist response of a4b2 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes (Peng et al. 1994;Hsu et al. 1996;Olale et al. 1997;Kuryatov et al. 2000). For example, incubations in 0.05-10 lM nicotine for 1-6 days reduce the response of a4b2 nAChRs expressed in Xenopus oocytes by 40-80% for a period of 24-48 h after the incubation ends (Peng et al. 1994;Hsu et al. 1996;Olale et al. 1997;Kuryatov et al. 2000). The molecular mechanism that underlies this persistent depression has not been established. However, it is thought to involve some kind of post-translational modification of the receptor (reviewed in Ochoa et al. 1989). It does not require persistent nicotine binding because [ 3 H]nicotine dissociates from rat brain nAChRs with a time constant of 2.5 min at 22°C (Lippiello et al. 1987).In contrast to oocytes, incubation in 0.1-10 lM nicotine for 1-7 days does not persistently inhibit the agonist response of a4b2 nAChRs expressed in mammalian cell lines (Gopalakrishnan et al. 1996;Buisson and Bertrand 2001). For example, a 7-day incubation in 100 nM nicotine increases the maximum ACh-induced 86 Rb influx from HEK cells expressing a4b2 nAChRs by 45% (Gopalakrishnan et al. 1996), and a 24-h incubation in 10 lM nicotine increases the maximum voltage-clamped ACh response of HEK cells expressing a4b2 nAChRs by 63% (Buisson and Bertrand 2001). Consistent with these increases, twice daily injections of nicotine tartrate (2 mg/kg) for 10 days 3 H]nicotine concentration at time t; NR, peak post-incubation a4b2 ACh response normalized to the pre-incubation value; NR 1 , final steady-state normalized response for recovery from ACh-induced desensitization; P solute , solute permeability coefficient; P nicotine , P solute for nicotine; t, time; s D , time constant for diffusion between two compartments; V cell , intracellular volume.

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Section: Xenopus Oocytes Accumulate and Release [ 3 H]nicotinementioning

confidence: 97%

Section: A 12‐h Incubation In 300 µM Ach Does Not Persistently Depresmentioning

confidence: 99%

Nicotine trapping causes the persistent desensitization of α4β2 nicotinic receptors expressed in oocytes

Flotildes

et al. 2003

Journal of Neurochemistry

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“…This motivated the development of 'Stopped Flow ESI-MS' by Kolakowski and Konermann in 2000 [9,10]. In 2003, Wilson and Konermann introduced a continuous flow capillary mixer for TR ESI-MS that facilitated full time-course acquisitions on time-scales comparable to optical stopped flow experiments [3].…”

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confidence: 99%

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

Rob

Wilson

2009

J. Am. Soc. Mass Spectrom.

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An electrospray coupled microfluidic reactor for the measurement of millisecond time-scale, solution phase kinetics is introduced. The device incorporates a simple two-channel design that is etched into polymethyl methacrylate (PMMA) by laser ablation. The outlet of the device is laser cut to a sharp tip, facilitating low dead volume 'on chip' electrospray. Fabrication is fast, straightforward and highly reproducible, supporting rapid prototyping and large-scale reproduction. Device performance is characterized using a cytochrome c unfolding reaction. Unfolding processes with rates in excess of 30 s Ϫ1 are easily measured, including the appearance of a 'native-like' intermediate that is maximally populated 180 ms post reaction initiation. To extract reliable rates from the data, a theoretical framework for the analysis of kinetics acquired under square-channel laminar flow is introduced. (J Am Soc Mass Spectrom 2009, 20, 124 -130)

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“…For reactions occurring within seconds or less, special instrumental setups have been designed based on the mixing of the species before MS measurement [23][24][25][26]. In our case, the subunit exchange was extremely slow: subunit exchange was observed at 37°C during more than 2 days without apparent degradation or loss of enzyme stability.…”

Section: Glms Subunit Exchange Occurs On Day Time Scalementioning

confidence: 99%

Monitoring the Dynamics of Monomer Exchange Using Electrospray Mass Spectrometry: The Case of the Dimeric Glucosamine-6-Phosphate Synthase

Chevreux

Atmanène

Lopez

et al. 2011

J. Am. Soc. Mass Spectrom.

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Escherichia coli glucosamine-6-phosphate synthase (GlmS) is a dimeric enzyme from the glutamine-dependent amidotransferases family, which catalyses the conversion of D-fructose-6-phosphate (Fru6P) and glutamine (Gln) into D-glucosamine-6-phosphate (GlcN6P) and glutamate, respectively. Extensive X-ray crystallography investigations have been reported, highlighting the importance of the dimeric association to form the sugar active site as well as significant conformational changes of the protein upon substrate and product binding. In the present work, an approach based on time-resolved noncovalent mass spectrometry has been developed to study the dynamics of GlmS subunit exchange. Using 14 N versus 15 N labeled proteins, the kinetics of GlmS subunit exchange was monitored with the wild-type enzyme in the presence of different substrates and products as well as with the protein bearing a key amino acid mutation specially designed to weaken the dimer interface. Determination of rate constants of subunit exchange revealed important modifications of the protein dynamics: while glutamine, glutamate, and K603A mutation accelerates subunit exchange, Fru6P and GlcN6P totally prevent it. These results are described in light of the available structural information, providing additional useful data for both the characterization of GlmS catalytic process and the design of new GlmS inhibitors. Finally, time-resolved noncovalent MS can be proposed as an additional biophysical technique for real-time monitoring of protein dynamics.

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Stopped-flow electrospray ionization mass spectrometry: a new method for studying chemical reaction kinetics in solution

Cited by 43 publications

References 23 publications

Nicotine trapping causes the persistent desensitization of α4β2 nicotinic receptors expressed in oocytes

Nicotine trapping causes the persistent desensitization of α4β2 nicotinic receptors expressed in oocytes

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

Monitoring the Dynamics of Monomer Exchange Using Electrospray Mass Spectrometry: The Case of the Dimeric Glucosamine-6-Phosphate Synthase

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