From Small-Molecule Reactions to Protein Folding: Studying Biochemical Kinetics by Stopped-Flow Electrospray Mass Spectrometry

Kolakowski, Beata; Konermann, Lars

doi:10.1006/abio.2001.5062

Cited by 35 publications

(27 citation statements)

References 48 publications

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“…The complementary advantages of microfluidic liquid handling and ESI-MS based analysis make these devices a natural answer to the challenges of proteomic studies. One 'underrepresented' application is rapid microfluidic mixing for time-resolved studies by ESI-MS. Time-resolved ESI-MS (TR ESI-MS) is a powerful approach for the characterization of shortlived, 'transient' intermediates in biochemical reactions [3][4][5][6][7][8][9][10], which are critical to the biological activity of proteins, from enzymatic reactions [7,11] to protein folding [12,13].…”

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confidence: 99%

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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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confidence: 99%

“…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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“…Proteins under native conditions show narrow charge state distributions in mass spectra with a small number of charges, whereas those under denaturing conditions tend to have broader charge state distribution with a higher number of charges. Thus, one can monitor protein conformations present in solution at equilibrium, and also kinetically during folding with time-resolved ESI-MS [7,8].…”

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confidence: 99%

Indirect assessment of small hydrophobic ligand binding to a model protein using a combination of ESI MS and HDX/ESI MS

Kaltashov

Eyles

2003

J. Am. Soc. Mass Spectrom.

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Direct mass spectrometric characterization of interactions between proteins and small hydrophobic ligands often poses a serious problem due to the complex instability in the gas phase. We have developed a method that probes the efficacy of ligand-protein interactions indirectly by monitoring changes in protein flexibility. The latter is assessed quantitatively using a combination of charge state distribution analysis and amide hydrogen exchange under both native and mildly denaturing conditions. The method was used to evaluate binding of a model protein cellular retinoic acid binding protein I to its natural ligand all-trans retinoic acid (RA), isomers 13-cis-and 9-cis-RA, and retinol, yielding the following order of ligand affinities: All-trans RA Ͼ 9-cis RA Ͼ 13-cis RA, with no detectable binding of retinol. This order is in agreement with the results of earlier fluorimetric titration studies. Furthermore, binding energy of the protein to each of retinoic acid isomers was determined based on the measured hydrogen exchange kinetics data acquired under native conditions. (J Am Soc Mass Spectrom 2003, 14, 506 -515)

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“…On-line applications have been carried out by effectively coupling home-built or commercial mixing devices to mass spectrometers equipped either with MIMS and analogous techniques (Degn & Kristensen, 1986;Northrop & Simpson, 1998;, or API sources (Sam, Tang, & Peisach, 1994;Sam et al, 1995;Kim et al, 1996;Konermann, Collings, & Douglas, 1997;Paiva et al, 1997;Zechel et al, 1998;Lee, Chen, & Konermann, 1999;Sogbein, Simmons, & Konermann, 1999;Kolakowski, Simmons, & Konermann, 2000;Kolakowski & Konermann, 2001). In seminal work using an API interface, a dual syringe pump was used to simultaneously drive two syringes connected to a low dead volume mixing tee; in turn, the tee was connected to the ionspray source of a triple quadrupole analyzer through a short section of fused silica capillary, which effectively served as reactor (Sam, Tang, & Peisach, 1994;Sam et al, 1995).…”

Section: Rapid Mixingmentioning

confidence: 99%

“…(Adapted from Sam, Tang, & Peisach, 1994. ) (Kim et al, 1996), to study the kinetics of protein folding and observe the transition between different discrete states in the folding process (Konermann, Collings, & Douglas, 1997;Sogbein, Simmons, & Konermann, 1999), and to perform pre-steady-state analysis of enzymatic pathways (Zechel et al, 1998;Kolakowski & Konermann, 2001;Konermann & Douglas, 2002). A dual syringe mixing system with ESI-MS allowed the observation of the presteady-state formation of a covalent intermediate between Bacillus circulans xylanase and the substrate 2,5-dinitrophenyl b-D-xylobioside (Zechel et al, 1998).…”

Section: Rapid Mixingmentioning

confidence: 99%

Mass spectrometric approaches for the investigation of dynamic processes in condensed phase

Fabris

2004

Mass Spectrometry Reviews

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Mass spectrometry (MS) offers many advantages over other established spectroscopic techniques employed for the investigation of processes in condensed phase. The sensitivity, specificity, and speed afforded by MS-based methods enable to obtain very valuable insights into the mechanism of complex dynamic processes. Off-line methods rely on quenching to halt the progress of the reaction of interest and allow for the implementation of a broad range of analytical procedures for sample fractionation, isolation, or desalting. On the contrary, on-line methods are designed to carry out the real-time monitoring of dynamic processes through a continuous uninterrupted analysis of reaction mixtures, with the only caveat that the sample solutions be directly amenable to the available ionization technique. The utilization of rapid mixing devices in direct connection with a mass spectrometer or included in off-line schemes provides access to the initial moments of a reaction, which can offer very important information about the reaction mechanism. This report summarizes the different off- and on-line strategies developed to study chemical and biochemical reactions in solution and obtain kinetic/mechanistic information. The merits of the various experimental designs, the characteristics of the different instrumental setups, and the factors affecting time resolution are discussed with the aid of specific examples, which highlight the contributions of MS to the different facets of the investigation of dynamic processes in condensed phase.

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From Small-Molecule Reactions to Protein Folding: Studying Biochemical Kinetics by Stopped-Flow Electrospray Mass Spectrometry

Cited by 35 publications

References 48 publications

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

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

Indirect assessment of small hydrophobic ligand binding to a model protein using a combination of ESI MS and HDX/ESI MS

Mass spectrometric approaches for the investigation of dynamic processes in condensed phase

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