Conformation of Cytochrome c Studied by Deuterium Exchange-Electrospray Ionization Mass Spectrometry

Wagner, David S.; Anderegg, Robert J.

doi:10.1021/ac00077a020

Cited by 121 publications

(147 citation statements)

References 19 publications

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“…These observations are in line with the well known fact that the ESI charge state distribution represents a sensitive probe of protein conformational changes. It is generally accepted that protein unfolding in solution leads to the formation of more highly charged protein ions in positive-ion ESI [77][78][79][80][81][82][83]. However, the physical basis of this phenomenon is still a matter of debate [23,84,85].…”

Section: Stokes Radiimentioning

confidence: 99%

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

Clark

Konermann

2003

J. Am. Soc. Mass Spectrom.

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This study describes a novel approach for monitoring noncovalent interactions in solution by electrospray mass spectrometry (ESI-MS). The technique is based on measurements of analyte diffusion in solution. Diffusion coefficients of a target macromolecule and a potential low molecular weight binding partner are determined by measuring the spread of an initially sharp boundary between two solutions of different concentration in a laminar flow tube (Taylor dispersion), as described in Rapid Commun. Mass Spectrom. 2002Spectrom. , 16, 1454Spectrom. -1462. In the absence of noncovalent interactions, the measured ESI-MS dispersion profiles are expected to show a gradual transition for the macromolecule and a steep transition for the low molecular weight compound. However, if the two analytes form a noncovalent complex in solution the dispersion profiles of the two species will be very similar, since the translational diffusion of the small compound is determined by the slow Brownian motion of the macromolecule. In contrast to conventional ESI-MS-based techniques for studying noncovalent complexes, this approach does not rely on the preservation of solution-phase interactions in the gas phase. On the contrary, "harsh" conditions at the ion source are required to disrupt any potential gasphase interactions between the two species, such that their dispersion profiles can be monitored separately. The viability of this technique is demonstrated in studies on noncovalent heme-protein interactions in myoglobin. Tight noncovalent binding is observed in solutions of pH 10, both in the absence and in the presence of 30% acetonitrile. In contrast, a significant disruption of the noncovalent interactions is seen at an acetonitrile content of 50%. Under these conditions, the diffusion coefficient of heme in the presence of myoglobin is only slightly lower than that of heme in a protein-free solution. A breakdown of the noncovalent interactions is also observed in aqueous solution of pH 2.4, where myoglobin is known to adopt an acid-unfolded conformation. (J Am Soc Mass Spectrom 2003, 14, 430 -441)

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Section: Stokes Radiimentioning

confidence: 99%

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

Clark

Konermann

2003

J. Am. Soc. Mass Spectrom.

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“…All species will exhibit the same HDX kinetics if interconversion is rapid on the HDX timescale. This relationship was first proposed in a seminal study by Wagner and Anderegg [53]. In that work it was reported that cytochrome c (cyt c) under mildly acidic conditions exhibits a bimodal charge-state distribution, representing the presence of unfolded proteins in addition to the native conformers.…”

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confidence: 94%

“…A key question that was not explicitly addressed in Wagner and Anderegg's study [53] and in related previous work [55] is why heat exposure of cyt c results in the formation of conformers that are irreversibly unfolded. The reasons underlying the apparently different HDX characteristics for high-and low-charge states thus remain unclear.…”

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confidence: 94%

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Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

Liu

Konermann

2009

J. Am. Soc. Mass Spectrom.

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This work uses electrospray ionization mass spectrometry (ESI-MS) in conjunction with hydrogen/deuterium exchange (HDX) and optical spectroscopy for characterizing the solutionphase properties of cytochrome c (cyt c) after heat exposure. Previous work demonstrated that heating results in irreversible denaturation for a subpopulation of proteins in the sample. However, that study did not investigate the physical reasons underlying this interesting effect. Here we report that the formation of oxidative modifications at elevated temperature plays a key role for the observed behavior. Tryptic digestion followed by tandem mass spectrometry is used to identify individual oxidation sites. Trp59 and Met80 are among the modified amino acids. In native cyt c both of these residues are buried deep within the protein structure, such that covalent modifications would be expected to be particularly disruptive. ESI-MS analysis after heat exposure results in a bimodal charge-state distribution. Oxidized protein appears predominantly in charge states around 11ϩ, whereas a considerably lower degree of oxidation is observed for the 7ϩ and 8ϩ peaks. This finding confirms that different oxidation levels are associated with different solution-phase conformations. HDX measurements for different charge states are complicated by peak distortions arising from oxygen adduction. Nonetheless, comparison with simulated peak shapes clearly shows that the HDX properties are different for high-and low-charge states, confirming that interconversion between unfolded and folded conformers is blocked in solution. In addition to oxidation, partial aggregation upon heat exposure likely contributes to the formation of irreversibly denatured protein. A number of well-established tools are available for experiments of this kind, including X-ray crystallography, nuclear magnetic resonance, calorimetry, and various spectroscopic techniques. Electrospray ionization mass spectrometry (ESI-MS) has become another widely used approach for exploring the properties of proteins in solution, providing information complementary to that obtained by traditional methods [1]. The ESI process generates intact gas-phase ions from proteins in solution. In the commonly used positive-ion mode these [M ϩ nH] nϩ species are multiply charged because of proton attachment.ESI of natively folded proteins results in protonation states n similar to those predicted for water droplets of the same size that are charged to the Rayleigh limit. This finding supports the idea that ionization follows the charged-residue model [2-6], although alternative scenarios have been proposed as well [7][8][9]. Much higher protonation states and wider charge-state distributions are generally seen for proteins that are unfolded in solution. Based on this empirical relationship, ESI-MS is now routinely being used for monitoring structural transitions in response to changes in pH, temperature, organic co-solvents, or covalent modifications [1, 10 -16]. The physical basis for the striking dependence of...

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“…[7][8][9][10][11] For example, laser-induced thermal desorption/Fourier-transform mass spectrometry has been applied to monitor the H/D exchange between ethylene and hydrogen (or deuterium) on Pt(111) surfaces. 12 Furthermore, H/D exchange kinetics has been used as a chemical probe for three-dimensional gaseous polypeptide ion structures.…”

Section: Introductionmentioning

confidence: 99%

Analysis of Self-Assembled Monolayers by Thermal Desorption Mass Spectrometry: Neighborhood Interaction and Hydrogen/Deuterium Exchange

Shibue

2004

ANAL. SCI.

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Conformation of Cytochrome c Studied by Deuterium Exchange-Electrospray Ionization Mass Spectrometry

Cited by 121 publications

References 19 publications

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

Analysis of Self-Assembled Monolayers by Thermal Desorption Mass Spectrometry: Neighborhood Interaction and Hydrogen/Deuterium Exchange

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