On the conformation‐dependent neutralization theory and charging of individual proteins and their non‐covalent complexes in the gas phase

Nesatyy, Victor J.; Suter, Marc J.‐F.

doi:10.1002/jms.522

Cited by 51 publications

(68 citation statements)

References 49 publications

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“…This finding supports the idea that ionization follows the charged-residue model [2][3][4][5][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.…”

supporting

confidence: 82%

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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supporting

confidence: 82%

Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

Liu

Konermann

2009

J. Am. Soc. Mass Spectrom.

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“…Excellent agreement between the predicted and observed maximum charge state of 79 proteins and noncovalent complexes was demonstrated by Nesatyy and Suter [37]. Table 3 [25] and Gross et al [2].…”

Section: Resultssupporting

confidence: 55%

Investigation of deprotonation reactions on globular and denatured proteins at atmospheric pressure by ESSI-MS

Touboul

Jecklin

Zenobi

2008

J. Am. Soc. Mass Spectrom.

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Deprotonation reactions of multiply charged protein ions have been studied by introducing volatile reference bases at atmospheric pressure between an electrosonic spray ionization (ESSI) source and the inlet of a mass spectrometer. Apparent gas-phase basicities (GB app ) of different charge states of protein ions were determined by a bracketing approach. The results pp obtained depend on the conformation of the protein ions in the gas phase, which is linked to the type of buffer used (denaturing or nondenaturing [1] rapidly became established as the method of choice for the production of large biomolecular ions in the gas phase. For proteins, ESI leads to the formation of multiply charged ions, both in the positive and negative ion modes. The charge-state distribution is directly related to the conformation of the macromolecular ions [2] and the proton transfer reactions in the gas phase [3]. Thermodynamic information, such as values for the gas-phase acidity/basicity (GA/GB), is essential to the understanding of the proton transfer processes and generation of ions by ESI in general. The intrinsic gas-phase basicity of a [M ϩ (n Ϫ 1)H] (n Ϫ 1)ϩ ion or the gas-phase acidity of a [M ϩ nH] nϩ ion can be defined as the free enthalpy change ⌬G 0 of the reaction:Since this reaction proceeds with a reverse activation barrier, due to Coulombic repulsion between the two charges product ions, the terms "apparent gas-phase acidity" (GA app ) and "apparent gas-phase basicity" (GB app ) are more appropriate. The GA app is defined as the sum of the ⌬G and the energy of the activation barrier, considered as a negative term [4].Determination of GB app s of peptides and proteins provides important information which is analogous to measurements of pKa in solution. For example, pKa values are greatly affected by intramolecular interactions, and the conversion of the denatured to the native form maximizes these interactions. Similarly, GB app values will also reflect the conformation of the ions in the gas phase.For more than 10 years, research groups have tried to determine apparent gas-phase basicities of peptides and proteins. For small peptides, like bradykinin and its des-arginine analogues, the kinetic method can be directly used to determine GB app values [5,6]. The energy of the activation barrier was obtained by measuring the kinetic energy release to calculate the intrinsic GB of two different charge states. Unfortunately, this method is quite difficult to transfer to multiply charged protein ions.A bracketing technique, consisting of evaluating the qualitative or quantitative rate of proton transfer between a neutral volatile base and protein ions was introduced to measure GB app values of multiply charges ions [4,7,8]. In terms of instrumentation, different systems can be used to observe the deprotonation reactions. High-pressure mass spectrometry (HPMS) experiments were designed by the group of Kebarle to study cluster formation from small organic or inorganic ions, and water in particular [9,10]. This apparatus was...

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“…In an attempt to further test the limiting model, Nesatyy°and°Suter° [29]°examined°a°much°larger°number of proteins and found that the agreement with the observed charge state was poor. They considered this as a proof against the CDN model especially since they found good agreement of the observed charges with the CRM°model° [9].…”

Section: The Cdn Model Of Grandori and Coworkers For Formation Of Mulmentioning

confidence: 99%

“…However,°neither°Grandori°[27a,°27b)]°nor°Nesatyy and°Suter° [29]°considered°using°X-ray°or°NMR°infor-mation to determine the number of salt bridges and their location in the various proteins. Such information is essential for estimating the number of ion pairs in solution because the buried salt bridges provide the most convincing case for acidic sites that are protected from neutralization.…”

Section: The Cdn Model Of Grandori and Coworkers For Formation Of Mulmentioning

confidence: 99%

Ion-Ion and ion-molecule reactions at the surface of proteins produced by nanospray. Information on the number of acidic residues and control of the number of ionized acidic and basic residues

Verkerk

Kebarle

2005

J. Am. Soc. Mass Spectrom.

102

150

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On the conformation‐dependent neutralization theory and charging of individual proteins and their non‐covalent complexes in the gas phase

Cited by 51 publications

References 49 publications

Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

Investigation of deprotonation reactions on globular and denatured proteins at atmospheric pressure by ESSI-MS

Ion-Ion and ion-molecule reactions at the surface of proteins produced by nanospray. Information on the number of acidic residues and control of the number of ionized acidic and basic residues

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