Quantitative evaluation of protein-protein and ligand-protein equilibria of a large allosteric enzyme by electrospray ionization time-of-flight mass spectrometry

Ayed, Ayeda; Krutchinsky, Andrew N.; Ens, Werner; Standing, Kenneth G.; Duckworth, Harry W.

doi:10.1002/(sici)1097-0231(19980415)12:7<339::aid-rcm163>3.0.co;2-6

Cited by 109 publications

(89 citation statements)

References 6 publications

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“…It is now widely accepted that a combination of collisional dampening, increased cooling of the ions, and more efficient desolvation is effective in the enhanced detection of ions with high m/z values. The fact that the detection of ions is m/z dependent makes it evident that this pressure effect phenomenon should be carefully addressed when relating ion abundance in mass spectra to solution-phase abundance of non-covalent protein assemblies, especially in equilibrium measurements between different components (Ayed et al, 1998).…”

Section: A Collisional Cooling And/or Focusingmentioning

confidence: 99%

Investigation of intact protein complexes by mass spectrometry

Heck

Heuvel

2004

Mass Spectrometry Reviews

553

554

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I. Introduction 00 II. Electrospray Ionization of Biomacromolecules 00 III. Mass Analyzers for Biomacromolecular Mass Spectrometry 00 A. Collisional Cooling and/or Focusing 00 B. Alternative Mass Analyzers 00 IV. Solution‐Phase Properties of Proteins Complexes 00 A. Protein–Protein Interactions 00 B. Cooperativity and Cofactors 00 C. Dynamics of Assembly 00 V. Gas‐Phase Properties of Protein Complexes 00 A. Tandem Mass Spectrometry 00 B. Charge Separation in Protein Dissociation 00 VI. Future Outlook 00 Acknowledgments 00 References 00 Mass spectrometry has grown in recent years to a well‐accepted and increasingly important complementary technique in structural biology. Especially electrospray ionization mass spectrometry is well suited for the detection of non‐covalent protein complexes and their interactions with DNA, RNA, ligands, and cofactors. Over the last decade, significant advances have been made in the ionization and mass analysis techniques, which makes the investigation of even larger and more heterogeneous intact assemblies feasible. These technological developments have paved the way to study intact non‐covalent protein–protein interactions, assembly and disassembly in real time, subunit exchange, cooperativity effects, and effects of cofactors, allowing us a better understanding of proteins in cellular processes. In this review, we describe some of the latest developments and several highlights. © 2004 Wiley Periodicals, Inc., Mass Spec Rev

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Section: A Collisional Cooling And/or Focusingmentioning

confidence: 99%

Investigation of intact protein complexes by mass spectrometry

Heck

Heuvel

2004

Mass Spectrometry Reviews

553

554

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“…One of the tools we investigate to measure protein aggregation involves using electrospray (ES) to generate vapor-phase dispersed material. These vapor-phase dispersed materials can then be analyzed by ion mobility methods named differential mobility analyzer (DMA), also known as gas-phase electrophoretic molecular analysis (GEMMA; Kaufman et al 1996;Bacher et al 2001;Kim and Zachariah 2005, 2007Pease et al 2008) or mass spectrometry (MS; Light-Wahl et al 1993Ayed et al 1998;Nettleton et al 2000;Lane et al 2009) methods. These techniques have the potential to characterize the distribution of oligomeric protein species in solution.…”

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confidence: 99%

Quantification and Compensation of Nonspecific Analyte Aggregation in Electrospray Sampling

Guha

Zangmeister

et al. 2011

Aerosol Science and Technology

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Electrospray (ES) sources are commonly used to introduce nonvolatile materials (e.g., nanoparticles, proteins, etc.) to the gas phase for characterization by mass spectrometry or ion mobility. Recent studies in our group using ES ion mobility to characterize protein aggregation in solution have raised the question as to whether the ES itself induces aggregation and thus corrupts the results. In this article, we develop a statistical model to determine the extent to which the ES process induces the formation of dimers and higher-order aggregates. The model is validated through ES differential mobility experiments using gold nanoparticles. The results show that the extent of droplet-induced aggregation is quite severe and previously reported cutoff criterion is inadequate. We use the model in conjunction with experiment to show the true dimer concentration in a protein solution as a function of concentration. The model is extendable to any ES source analytical system and to higher aggregation states. For users only interested in implementation of the theory, we provide a section that summarizes the relevant formulas.

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“…Although these studies have been extended to quantitative measurements of binding affinities of noncovalent complexes [2][3][4], it is still not clear to what extent the relative yield of gas-phase ions produced by ESI represents their relative solution concentrations. If electrospray ionization mass spectrometry (ESI-MS) is to be used to determine binding constants of noncovalent complexes, this question is of profound significance.…”

mentioning

confidence: 99%

Shrinking droplets in electrospray ionization and their influence on chemical equilibria

Wortmann

Kistler-Momotova

Zenobi

et al. 2007

J. Am. Soc. Mass Spectrom.

115

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We investigated how chemical equilibria are affected by the electrospray process, using simultaneous in situ measurements by laser-induced fluorescence (LIF) and phase Doppler anemometry (PDA). The motivation for this study was the increasing number of publications in which electrospray ionization mass spectrometry is used for binding constant determination. The PDA was used to monitor droplet size and velocity, whereas LIF was used to monitor fluorescent analytes within the electrospray droplets. Using acetonitrile as solvent, we found an average initial droplet diameter of 10 m in the electrospray. The PDA allowed us to follow the evolution of these droplets down to a size of 1 m. Rhodamine B-sulfonylchloride was used as a fluorescent analyte within the electrospray. By spatially resolved LIF it was possible to probe the dimerization equilibrium of this dye. Measurements at different spray positions showed no influence of the decreasing droplet size on the monomer-dimer equilibrium. However, with the fluorescent dye pair DCM and oxazine 1 it was shown that a concentration increase does occur within electrosprayed droplets, using fluorescence resonance energy transfer as a probe for the average pair distance. ( An important aspect of the highly dynamic electrospray process concerns the shrinking droplets. After the droplet creation, solvent evaporation and Coulomb explosions lead to a decrease of droplet size until all solvent has evaporated and the final desolvated and ionized analytes are produced. A decrease of droplet size resulting from solvent evaporation will lead to a concentration increase of nonvolatile analytes within the droplets. For dissolved species that participate in a chemical equilibrium, such as free and bound states of partners that can form a noncovalent complex, the question arises whether the increasing concentrations affect the equilibrium position. Given that binding constant determination with ESI-MS usually relies on measuring the relative amounts of free and bound species, a perturbation of the equilibrium introduced by the shrinking droplets would influence the results of such an experiment. Recent studies show that the time between droplet creation and production of desolvated ions in ESI is within milliseconds [5]. In other words, if the association and dissociation reactions are fast, an equilibrium should be able to track a concentration increase within the droplets.The shrinking droplet volume also leads to Coulomb explosions/droplet fission, when the mutual repulsion of charges is high enough to overcome the surface tension of the droplet. The parent droplet ejects several smaller offspring droplets, which together carry 10 -25% of the charge, but only about 5% of the mass of the original droplet [9]. These offspring droplets then segregate within the electrospray, as shown theoretically by Wilhelm et al. [10]. Analytes will not generally be evenly distributed between the parent and offspring droplets. Partitioning processes, which have been investigated by several research g...

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Quantitative evaluation of protein-protein and ligand-protein equilibria of a large allosteric enzyme by electrospray ionization time-of-flight mass spectrometry

Cited by 109 publications

References 6 publications

Investigation of intact protein complexes by mass spectrometry

Investigation of intact protein complexes by mass spectrometry

Quantification and Compensation of Nonspecific Analyte Aggregation in Electrospray Sampling

Shrinking droplets in electrospray ionization and their influence on chemical equilibria

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