Probing conformational changes in proteins by mass spectrometry

Chowdhury, Swapan K.; Katta, Viswanatham; Chait, Brian T.

doi:10.1021/ja00180a074

Cited by 632 publications

(684 citation statements)

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“…37 However, an important factor governing the charge state distribution in the MS-spectrum is the conformation of the protein in solution. 38,39 The observation of only a few peaks, serves as evidence for a folded (native) protein, while the observation of broadly distributed charge states of high charge is an indication of denaturation. 29 From these observations we conclude that lysozyme in this study was not significantly unfolded in any of the experiments.…”

Section: Inmentioning

confidence: 99%

Interfacial Complexes between a Protein and Lipophilic Ions at an Oil−Water Interface

et al. 2010

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The interaction between an intact protein and two lipophilic ions at an oil-water interface has been investigated using cyclic voltammetry, impedance based techniques and a newly developed method in which the biphasic oil-water system is analyzed by biphasic electrospray ionization mass spectrometry (BESI-MS), using a dualchannel electrospray emitter. It is found that the protein forms interfacial complexes with the lipophilic ions and that it specifically requires the presence of the oil-water interface to be formed under the experimental conditions. Furthermore, impedance based techniques and BESI-MS with a common ion to polarize the interface indicated that the Galvani potential difference across the oil-water interface significantly influences the interfacial complexation degree. The ability to investigate protein-ligand complexes formed at polarized liquid-liquid interfaces is thus a new analytical method for assessing potential dependent interfacial complexation using a structure elucidating detection principle.The behavior of biological macromolecules at liquid-liquid interfaces has important implications in the pharmaceutical sciences. It is for example well-known that proteins adsorb at a wide variety of interfaces, 1-4 a process that can have unwanted consequences such as a loss of therapeutic activity due to denaturation and aggregation. 4,5 Two-phase systems are at equilibrium generally associated with an interfacial potential difference, 6 in the following referred to as the Galvani potential difference between a water (w) and oil (o) phase at equilibrium; ∆ o w φ. The value of ∆ o w φ can influence adsorption kinetics and/ or adsorption isotherms. 7 Electrochemistry at Interfaces between two immiscible electrolyte solutions (ITIES) provides an effective methodology for studying potential dependent adsorption and ion transfer processes. [8][9][10][11][12] In relation to protein research, electrochemistry at ITIES have been used to study protein adsorption, 13 protein adsorption kinetics, 7 the effects on transfer of aqueous ions, 14 and the assisted transfer of proteins to the oil phase. [15][16][17][18] Recently, electrochemistry at ITIES has been used for detecting proteins in solution by means of a protein assisted transfer of organic anions into the water phase at positive potentials. [19][20][21][22] A suggested mechanism for the organic anion transfer involves the formation of a protein-anion complex at the interface. 22 Usually electrospray ionization mass spectrometry (ESI-MS) involves an aqueous solution which by application of a high voltage is sprayed into a mass analyzer. Recently, the development of a new biphasic electrospray interface has allowed interfacial complexes in two phase systems to be analyzed using a mass spectrometer; the new technique is termed biphasic electrospray mass spectrometry (BESI-MS). [23][24][25][26] The combined use of BESI-MS and electrochemistry at ITIES is attractive as it allows studying quantitative mechanistic aspects of adsorption and ion transfer pro...

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Section: Inmentioning

confidence: 99%

Interfacial Complexes between a Protein and Lipophilic Ions at an Oil−Water Interface

et al. 2010

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“…The mass spectra of proteins is characterised by a succession of peaks whose m/z corresponds to different charge states of the intact protein. It is accepted that the shape of the charged distribution does mainly depend on the availability of the various chargeable basic sites (Arg, Lys, His and the N-terminus) [8], and thus to the tertiary structure of the protein (i. e. in a highly folded protein few basic sites will be available, however, in a fully unfolded state all basic sites should be accessible) [9]. This effect on the charge state distribution of protein has been verified studying the influence of acids [10], temperature [11] or solvents [12].…”

Section: Introductionmentioning

confidence: 99%

Time of flight versus ion trap MS coupled to CE to analyse intact proteins

Erny¹,

León²,

Marina

et al. 2008

J of Separation Science

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In this work, two different CE-MS instruments, namely, CE-ESI-IT-MS and CE-ESI-TOF-MS, applied to analyse intact proteins from complex samples are investigated. The aim of this work was to compare both instruments in terms of LOD, number of proteins detected, and precision and repeatability in the determination of the protein relative molecular mass. Results show that although CE-ESI-IT-MS provides cleaner MS spectra of intact proteins, CE-ESI-TOF-MS allows the identification of a higher number of proteins from complex matrices in an easier way. Performance in terms of peak area reproducibility, LOD and precision in the determination of the molecular mass were similar for both instruments. The usefulness of the optimised CE-ESI-IT-MS and CE-ESI-TOF-MS conditions was demonstrated by studying the zein-proteins composition of three natural maize lines and their corresponding transgenic lines, showing no significant differences.

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“…They also observed that reducing the conformational flexibility of the proteins decreases the extent of asymmetric dissociation of the complex. Chowdhury et al [20] pointed out that multiply charged ions are produced primarily as a result of proton attachment to the available basic sites in the protein, and that the availability of ionizable basic sites depends upon the conformation of the protein. In general, a protein in an unfolded conformation may possess more available basic sites than those in tightly folded conformations.…”

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confidence: 99%

Theoretical investigations of the dissociation of charged protein complexes in the gas phase

Wanasundara

Thachuk

2007

J. Am. Soc. Mass Spectrom.

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A series of calculations, varying from simple electrostatic to more detailed semi-empirical based molecular dynamics ones, were carried out on charged gas phase ions of the cytochrome c dimer. The energetics of differing charge states, charge partitionings, and charge configurations were examined in both the low and high charge regimes. As well, preliminary free energy calculations of dissociation barriers are presented. It is shown that one must always consider distributions of charge configurations, once protein relaxation effects are taken into account, and that no single configuration dominates. All these results also indicate that in the high charge limit, the dissociation of protein complex ions is governed by electrostatic repulsion from the net charges, the consequences of which are enumerated and discussed. There are two main trends deriving from this, namely that charges will move so as to approximately maintain constant surface charge density, and that the lowest barrier to dissociation is the one that produces fragment ions with equal charges. In particular, it is shown that the charge-to-mass ratio of a fragment ion is not the key physical parameter in predicting dissociation products. In fact, from the perspective of the division of total charge, many dissociation pathways reported to be "asymmetric" in the literature should be more properly labelled as "symmetric" or "near-symmetric". The Coulomb repulsion model assumes that the timescale for charge transfer is faster than that for protein structural changes, which in turn is faster than that for complex dissociation. One challenge in using mass spectrometry for the general analysis of protein complexes is understanding the dissociation mechanism of these complexes in the gas phase. Many groups [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] have reported asymmetric dissociation behaviour for large multimeric proteins. A small subunit, typically a protein monomer, is ejected from the complex during dissociation, with the monomer carrying away a disproportionate amount of charge for its relative mass. Understanding the cause of this phenomenon will help to improve our understanding of the structural properties of noncovalent complexes.It is important to investigate how these dissociations occur and the factors that influence them. Using Fourier-transform mass spectrometry (FTMS), Jurchen and Williams [6] have conducted a number of studies in order to understand the origin of these charge distributions. In these experiments, isolated charge states of cytochrome c dimer were dissociated by sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD). According to Jurchen and Williams [6], the asymmetric charge distribution depends upon charge state, dissociation energy, and conformational flexibility. These studies showed that higher charge states lead to symmetric charge products while lower charge states lead to an asymmetric charge dissociation pathway. Further, their results with different excitation energies show ...

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Probing conformational changes in proteins by mass spectrometry

Cited by 632 publications

References 1 publication

Interfacial Complexes between a Protein and Lipophilic Ions at an Oil−Water Interface

Interfacial Complexes between a Protein and Lipophilic Ions at an Oil−Water Interface

Time of flight versus ion trap MS coupled to CE to analyse intact proteins

Theoretical investigations of the dissociation of charged protein complexes in the gas phase

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