Interpreting conformational effects in protein nano-ESI-MS spectra

Šamalíková, Mária; Matecko, Irena; Müller, Norbert; Grandori, Rita

doi:10.1007/s00216-003-2339-6

Cited by 73 publications

(113 citation statements)

References 55 publications

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“…The spectrum resembles those obtained for lysozyme solutions without an oil phase, obtained with the same spectrometer (data not shown) and in other laboratories. 36 The spectrum is dominated by two peaks at m/z ) 1432 and m/z ) 1591 corresponding to the +10 and +9 charge states of lysozyme, respectively. From hydrogen-ion titrations an overall positive charge of around 10 for lysozyme is expected in solution at the measured pH of 2.81 and low ionic strength.…”

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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“…Cytochrome c ions are quite sensitive to the mass spectrometric conditions. The group of Grandori proved that cytochrome c is prone to denaturation when increasing the curtain gas flow or the pH [44]. Due to the supersonic nebulizing gas used for ESSI measurements, partial denaturation is possible and a considerable number of salts bridges on the protein surface could be destroyed.…”

Section: Cytochrome Cmentioning

confidence: 99%

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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“…Also, a variety of factors may affect the results of the ESI-MS in studying protein conformational changes (Rockwood et al 1991;Mirza and Chait 1997;Hearn et al 2002;Samalikova et al 2004). We tested the effect of spray voltage and other ion source settings (nebulizer gas pressure, dry gas pressure and temperature, cap exit voltage, spray voltage, etc.)…”

Section: Lysozyme Unfolding Characterized By Esi-msmentioning

confidence: 99%

The first step of hen egg white lysozyme fibrillation, irreversible partial unfolding, is a two‐state transition

Shashilov

Ermolenkov

et al. 2007

Protein Science

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Amyloid fibril depositions are associated with many neurodegenerative diseases as well as amyloidosis. The detailed molecular mechanism of fibrillation is still far from complete understanding. In our previous study of in vitro fibrillation of hen egg white lysozyme, an irreversible partially unfolded intermediate was characterized. A similarity of unfolding kinetics found for the secondary and tertiary structure of lysozyme using deep UV resonance Raman (DUVRR) and tryptophan fluorescence spectroscopy leads to a hypothesis that the unfolding might be a two-state transition. In this study, chemometric analysis, including abstract factor analysis (AFA), target factor analysis (TFA), evolving factor analysis (EFA), multivariate curve resolutionalternating least squares (ALS), and genetic algorithm, was employed to verify that only two principal components contribute to the DUVRR and fluorescence spectra of soluble fraction of lysozyme during the fibrillation process. However, a definite conclusion on the number of conformers cannot be made based solely on the above spectroscopic data although chemometric analysis suggested the existence of two principal components. Therefore, electrospray ionization mass spectrometry (ESI-MS) was also utilized to address the hypothesis. The protein ion charge state distribution (CSD) envelopes of the incubated lysozyme were well fitted with two principal components. Based on the above analysis, the partial unfolding of lysozyme during in vitro fibrillation was characterized quantitatively and proven to be a two-state transition. The combination of ESI-MS and Raman and fluorescence spectroscopies with advanced statistical analysis was demonstrated to be a powerful methodology for studying protein structural transformations.

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Interpreting conformational effects in protein nano-ESI-MS spectra

Cited by 73 publications

References 55 publications

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

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

The first step of hen egg white lysozyme fibrillation, irreversible partial unfolding, is a two‐state transition

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