Quantitative determination of noncovalent binding interactions using soft ionization mass spectrometry

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)

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confidence: 98%

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confidence: 99%

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

Akashi

Osawa

Nishimura

2005

“…Unlike these techniques, MS is capable of resolving any free/bound species at equilibrium in solution, even when such species possess very similar spectroscopic characteristics. With proper experimental design and data treatment, their respective signal intensities can be employed to obtain relative [121,122] and absolute [123][124][125][126][127][128] dissociation constants (K d 's) in solution, matching those afforded by established methods [129]. In this direction, competitive binding experiments in which multiple ligands are mixed simultaneously with the substrate of interest have proven very effective in providing relative scales of binding affinities based on the aspect ratio and distribution of the detected complexes [130 -133].…”

Section: Elucidating Structure-function Relationshipsmentioning

confidence: 99%

A eole for the MS analysis of nucleic acids in the post-genomics age

Fabris

2010

The advances of mass spectrometry in the analysis of nucleic acids have tracked very closely the exciting developments of instrumentation and ancillary technologies, which have taken place over the years. However, their diffusion in the broader life sciences community has been and will be linked to the ever evolving focus of biomedical research and its changing demands. Before the completion of the Human Genome Project, great emphasis was placed on sequencing technologies that could help accomplish this project of exceptional scale. After the publication of the human genome, the emphasis switched toward techniques dedicated to the exploration of sequences not coding for actual protein products, which amount to the vast majority of transcribed elements. The broad range of capabilities offered by mass spectrometry is rapidly advancing this platform to the forefront of the technologies employed for the structure-function investigation of these noncoding elements. Increasing focus on the characterization of functional assemblies and their specific interactions has prompted a re-evaluation of what has been traditionally construed as nucleic acid analysis by mass spectrometry. Inspired by the accelerating expansion of the broader field of nucleic acid research, new applications to fundamental biological studies and drug discovery will help redefine the evolving role of MS-analysis of nucleic acids in the post-genomics age. (J Am Soc Mass Spectrom 2010, 21, 1-13)

“…Four primary factors influence the observed dissociation energies of the polyether/ammonium ion complexes: the gas-phase basicities of the polyether and amine, steric effects of the amines, conformational flexibility of the polyethers, and the inhibition of intramolecular hydrogen bonds of the guest ammonium ions in the resulting ammonium/polyether noncovalent complexes. E lectrospray ionization mass spectrometry (ESI-MS) is being increasingly used for the analysis of noncovalent complexes, especially those involving biomolecules, because it is a "soft" method for the transfer of solution species into the gas phase [1][2][3][4][5]. A key question revolves around the nature of the noncovalent complexes once they enter the gas phase.…”

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confidence: 99%

Threshold dissociation energies of protonated amine/polyether complexes in a quadrupole ion trap

David

Brodbelt

2003

Electrospray ionization mass spectrometry (ESI-MS) is increasingly used to probe the nature of noncovalent complexes; however, assessing the relevance of gas-phase results to structures of complexes in solution requires knowledge of the types of interactions that are maintained in a solventless environment and how these might compare to key interactions in solution. This study addresses the factors impacting the strength of hydrogen bonding noncovalent interactions in the gas phase. Hydrogen bonded complexes consisting of ammonium ions bound to polydentate ethers are transported to the gas phase with ESI, and energy-variable collisional activated dissociation (CAD) is used to map the relative dissociation energies. The measured relative dissociation energies are correlated with the gas-phase basicities and steric factors of the amine and polyether constituents. To develop correlations between hydrogen bonding strength and structural features of the donor and acceptor molecules, a variety of amines with different gas-phase basicities and structures were selected, including primary, secondary, and tertiary amines, as well as those that are bidentate to promote intramolecular hydrogen bonding. The acceptor molecules are polydentate ethers, such as 18-crown-6. Four primary factors influence the observed dissociation energies of the polyether/ammonium ion complexes: the gas-phase basicities of the polyether and amine, steric effects of the amines, conformational flexibility of the polyethers, and the inhibition of intramolecular hydrogen bonds of the guest ammonium ions in the resulting ammonium/polyether noncovalent complexes. E lectrospray ionization mass spectrometry (ESI-MS) is being increasingly used for the analysis of noncovalent complexes, especially those involving biomolecules, because it is a "soft" method for the transfer of solution species into the gas phase [1][2][3][4][5]. A key question revolves around the nature of the noncovalent complexes once they enter the gas phase. The specificity and the type of interactions that are retained in a solventless environment are important considerations when assessing the relevance of gas-phase results to the structures of solution-phase complexes [1][2][3][4][5]. Information on the relative stabilities of noncovalent complexes is often obtained from their gas-phase dissociation behavior [6], and many recent examples of biologically interesting macromolecular complexes demonstrate the importance of electrostatic interactions and hydrogen bonds in maintaining these noncovalent associations in the gas phase [7][8][9][10][11][12][13][14]. In some cases, the stabilities of complexes obtained based on gas-phase measurements do not correlate well with those obtained in solution [14], whereas other reports have shown remarkable agreement between gas-phase and solution results [13].Hydrogen bonds are one of the most important types of noncovalent interactions in the gas phase, both in simple protonated molecules, such as peptides and proteins, and in large biological com...