Observation of the heme-globin complex in native myoglobin by electrospray-ionization mass spectrometry

Katta, Viswanatham; Chait, Brian T.

doi:10.1021/ja00022a058

Cited by 449 publications

(378 citation statements)

References 0 publications

Supporting

Mentioning

357

Contrasting

Unclassified

Order By: Relevance

“…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]. However, recently it has also been shown that proteins themselves can have an important influence on the response of other proteins [13].…”

Section: Introductionmentioning

confidence: 88%

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

Erny¹,

León²,

Marina

et al. 2008

J of Separation Science

View full text Add to dashboard Cite

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.

show abstract

Section: Introductionmentioning

confidence: 88%

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

Erny¹,

León²,

Marina

et al. 2008

J of Separation Science

View full text Add to dashboard Cite

show abstract

“…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

View full text Add to dashboard Cite

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...

show abstract

“…2 Gray boxes indicate the mutated positions in mutant dsDNA. 3 Relative binding free energy change associated with the base-pair substitution can be calculated by the following equation: ⌬⌬G ϭ ⌬Gm Ϫ ⌬Gwt ϭ Ϫ RT ln Kd͑m͒ Kd͑wt͒ ⌬Gm: ⌬G for mutant, ⌬Gwt: ⌬G for wild type (m22) Kd(m): Kd for mutant, Kd(wt): Kd for wild type (m22)Kd for each complex was determined by filter-binding assay. The DNA and c-Myb DBD was incubated in buffer (100mM potassium phosphate (pH7.5), 20mM KCl, 0.1mM EDTA, 500 g/mL of BSA, 5% glycerol (v/v), 10mM dithiothreitol) on ice, and then filtered through nitrocellulose under suction [11].…”

Section: Characterization Of the Complex Stability By Cidmentioning

confidence: 99%

“…Consequently, positive correlation was obtained between V 50% and relative binding free energy change (⌬⌬G) [1]. In addition, ESI-MS has allowed successful detection of noncovalent complexes of biomolecules [2][3][4][5][6]. Direct observation of molecular ions of noncovalent complexes is significant in understanding protein function, because it suggests a target, specificity, and stoichiometry of the specific binding.…”

mentioning

confidence: 99%

Evaluation of protein-DNA binding affinity by electrospray ionization mass spectrometry

Akashi

Osawa

Nishimura

2005

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

Binding affinity of complexes between a DNA-binding domain (DBD) of a transcription factor, c-Myb, and several double-stranded DNA (dsDNA) were evaluated by collision-induced dissociation (CID) of the multiply protonated molecules generated by electrospray ionization mass spectrometry (ESI-MS). Complexes of the c-Myb DBD and dsDNA were prepared in solution and analyzed by ESI-MS. Multiply protonated molecules of a high-affinity complex, the c-Myb DBD and dsDNA with a specific sequence, were clearly observed in ESI mass spectrum. Protonated molecules of the complex were quite stable in the gas-phase, and not easily dissociated even if high cone voltage was applied in the first vacuum chamber source when the sample was prepared in 10 mM ammonium acetate. As for the sample prepared in buffer with higher concentration of ammonium acetate, such as 500 mM ammonium acetate, protein-dsDNA complexes could easily be dissociated with an increase in the cone voltage, giving multiply protonated molecules of free c-Myb DBD and some DNA fragments. Systematic CID experiments were carried out on seven complexes between the c-Myb DBD and 22-mer dsDNA with different solution-Kd values in the range of 10 Ϫ9 M to 10 Ϫ7 M. For each complex dissociation curve as a function of cone voltage was plotted, and the cone voltage where 50% of the complex was dissociated (V 50% ) was calculated. Consequently, positive correlation was obtained between V 50% and relative binding free energy change (⌬⌬G) in complex formation in solution. This suggests that ESI-CID experiments can provide quantitative evaluation of the stability of protein-DNA complexes based on proper calibration. (J Am Soc Mass Spectrom 2005, 16, 116 -125)

show abstract

Observation of the heme-globin complex in native myoglobin by electrospray-ionization mass spectrometry

Cited by 449 publications

References 0 publications

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

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

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

Evaluation of protein-DNA binding affinity by electrospray ionization mass spectrometry

Contact Info

Product

Resources

About