Electrospray ionization mass spectrometry analysis revealed a ∼310 kDa noncovalent hexamer of HPr kinase/phosphatase from Bacillus subtilis

Sanglier, Sarah; Ramström, Helena; Haiech, Jacques; Leize, Emmanuelle; Dorsselaer, Alain Van

doi:10.1016/s1387-3806(02)00747-9

Cited by 17 publications

(15 citation statements)

References 49 publications

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“…Therefore, a careful selection of the MS transfer conditions, i.e., the transfer voltages and the gas pressure, is required where the internal energy of the ions is below the dissociation threshold of the complex (Sobott et al 2005). Experimentally, this is controlled by reducing the transfer voltages, which control the kinetic energy of the ions, and/or increasing the source pressure (Schmidt et al 2001;Tahallah et al 2001;Sanglier et al 2002;Tjernberg et al 2004). Thus, the detection of the folded hERa LBD monomer at a native pH could in theory be due to homodimer dissociation during the MS transfer.…”

Section: Detection Of Native Hera Lbd By Nanoesi-msmentioning

confidence: 99%

“…The cone and first ion tunnel RF1 voltages, the parameters that control the kinetic energy of the ions in the source region of the Q-ToF ULTIMA mass spectrometer, were optimized at 80 V and 60 V, respectively. The pressure in the source region was increased at 4.5 mbar with a Speedivalve (BOC Edwards) to enhance the transmission of high m /z ions (Tito et al 2001;Sobott et al 2002;Chernushevich and Thomson 2004) and to preserve the noncovalent complexes in the gas phase (Schmidt et al 2001;Tahallah et al 2001;Sanglier et al 2002;Tjernberg et al 2004). After passing the cone and the ion tunnels, the ion beam was transmitted to the quadrupole used in RF-only mode and passed through a hexapole collision cell pressurized with argon (Purity 5.0, PanGas).…”

Section: Electrospray Ionization Mass Spectrometrymentioning

confidence: 99%

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Estrogen receptor–ligand complexes measured by chip‐based nanoelectrospray mass spectrometry: An approach for the screening of endocrine disruptors

et al. 2007

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In the present report, a method based on chip-based nanoelectrospray mass spectrometry (nanoESI-MS) is described to detect noncovalent ligand binding to the human estrogen receptor a ligand-binding domain (hERa LBD). This system represents an important environmental interest, because a wide variety of molecules, known as endocrine disruptors, can bind to the estrogen receptor (ER) and induce adverse health effects in wildlife and humans. Using proper experimental conditions, the nanoESI-MS approach allowed for the detection of specific ligand interactions with hERa LBD. The relative gasphase stability of selected hERa LBD-ligand complexes did not mirror the binding affinity in solution, a result that demonstrates the prominent role of hydrophobic contacts for stabilizing ER-ligand complexes in solution. The best approach to evaluate relative solution-binding affinity by nanoESI-MS was to perform competitive binding experiments with 17b-estradiol (E2) used as a reference ligand. Among the ligands tested, the relative binding affinity for hERa LBD measured by nanoESI-MS was 4-hydroxtamoxifen % diethylstilbestrol > E2 >> genistein >> bisphenol A, consistent with the order of the binding affinities in solution. The limited reproducibility of the bound to free protein ratio measured by nanoESI-MS for this system only allowed the binding constants (K d ) to be estimated (low nanomolar range for E2). The specificity of nanoESI-MS combined with its speed (1 min/ligand), low sample consumption (90 pmol protein/ligand), and its sensitivity for ligand (30 ng/mL) demonstrates that this technique is a promising method for screening suspected endocrine disrupting compounds and to qualitatively evaluate their binding affinity.Keywords: electrospray ionization mass spectrometry; noncovalent; nuclear receptor; estrogen receptor; endocrine disruptors; solution affinityThe estrogen receptor (ER) belongs to the nuclear receptor (NR) superfamily of ligand-activated transcription regulators, which are involved in many processes such as growth, organ differentiation, and development of reproductive tissues. The ER, which comprises a DNA-binding domain (DBD) and a ligand-binding domain (LBD), activates the transcription of target genes in response to the binding of estrogens, a group of steroid compounds, to the LBD. Once bound by hormones, the ER undergoes a conformational change that facilitates dimerization and subsequent interactions with the specific DNA sequence (Kumar and Chambon 1988;Steinmetz et al. 2001). The large hydrophobic cavity of the ER LBD allows the binding of a wide variety of nonsteroidal compounds through hydrophobic interactions. In the last decades, a variety of Reprint requests to: Renato Zenobi, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland; e-mail: zenobi@org.chem.ethz.ch; fax: 41 44 632 12 92.Article published online ahead of print. Article and publication date are at

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Section: Detection Of Native Hera Lbd By Nanoesi-msmentioning

confidence: 99%

Section: Electrospray Ionization Mass Spectrometrymentioning

confidence: 99%

Estrogen receptor–ligand complexes measured by chip‐based nanoelectrospray mass spectrometry: An approach for the screening of endocrine disruptors

et al. 2007

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“…Previously, ESI MS has been used for evaluation of oligomers and prefibrillar aggregates of various peptides and proteins [51], such as, for example, amyloid-β peptide fragment 10–30 [52], nucleobase-functionalized beta-peptides [53], insulin [54], transthyretins [36,55,56], human beta-2-microglobulin [57], native hemocyanins [58,59], flavoproteins [60], bifunctional kinase/phosphatase [58]. We have previously used ESI MS for the determination of the complex between oligomers, tetramers, and dimers of stefin B and monomers of amyloid-β peptide [42].…”

Section: Introductionmentioning

confidence: 99%

“…Commonly, protein tetramers [55], hexamers [52,58] or octamers [60] have been observed, but higher-order oligomers like 18-mers [59,61] have also been detected. In most of the studies thus far, mainly even-numbered oligomers have been described, whereas in some cases, odd-numbered oligomers have been reported to occur [57].…”

Section: Introductionmentioning

confidence: 99%

The Role of Initial Oligomers in Amyloid Fibril Formation by Human Stefin B

Taler‐Verčič

Kirsipuu

Friedemann

et al. 2013

IJMS

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Oligomers are commonly observed intermediates at the initial stages of amyloid fibril formation. They are toxic to neurons and cause decrease in neural transmission and long-term potentiation. We describe an in vitro study of the initial steps in amyloid fibril formation by human stefin B, which proved to be a good model system. Due to relative stability of the initial oligomers of stefin B, electrospray ionization mass spectrometry (ESI MS) could be applied in addition to size exclusion chromatography (SEC). These two techniques enabled us to separate and detect distinguished oligomers from the monomers: dimers, trimers, tetramers, up to decamers. The amyloid fibril formation process was followed at different pH and temperatures, including such conditions where the process was slow enough to detect the initial oligomeric species at the very beginning of the lag phase and those at the end of the lag phase. Taking into account the results of the lower-order oligomers transformations early in the process, we were able to propose an improved model for the stefin B fibril formation.

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“…To preserve the fragile noncovalent protein complex, it is generally accepted that "gentle" conditions should be maintained by mild desolvation voltages and low temperature in the atmosphere-vacuum ESI interface conditions [21]. However, the detection of very large multi-protein complex represents extreme cases where high interface pressure and accelerating voltages are usually preferred [22]. Due to the mass-dependent ion transmission in the interface region and quadrupoles, the analysis of large multiprotein or multi-subunit assembly by ESI-MS still represents an analytical challenge which is far from routine operation.…”

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confidence: 99%

Structural characterization of sialic acid synthase by electrospray mass spectrometry—A tetrameric enzyme composed of dimeric dimers

Huang

Liao

Chen

et al. 2005

J. Am. Soc. Mass Spectrom.

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Sialic acid synthase (NeuB) encoded by the neuB gene catalyzes the condensation of N-acetylmannosamine and phospho(enol)pyruvate to form N-acetylneuraminic acid. The enzyme is essential for the biosynthesis of polysialic acid, a capsular sugar polymer functioning as a virulent factor and antiphagocytic barrier. This report demonstrates the first characterization on the quaternary structure of NeuB from Escherichia coli (EcNeuB) and Streptococcus agalactiae (SaNeuB) by nanoflow electrospray ionization mass spectrometry (ESI-MS). Under non-denaturing conditions, Tris buffer was observed to induce a higher ratio of tetramer/dimer of NeuB in the ESI mass spectra, providing supportive evidence for the existence of a "structurally-specific" tetramer. The instrument parameters were found to significantly affect the ratio of detected tetramer/dimer in ESI mass spectra. The harshest conditions, including high desolvation voltages and pressure in the collision cell, led to enhanced detection of the 160 kDa tetramer. The prevalence of dimeric form is likely the cause in loss of tetramer stability in gas-phase arising from insufficient collisional cooling, which implies an asymmetric assembly, possibly composed of dimeric dimers. Most interestingly, the hypothesis was further supported by chemical cross-linking of SaNeuB, in which the reaction of shorter linker yielded mainly the dimer whereas that of longer linker produced both dimer and tetramer. Furthermore, the ESI-MS analysis can reflect dramatic change of pH-dependent quaternary structure in association with enzyme activity, suggesting the tetrameric form may be the primary species responsible for the enzyme catalysis. (J Am Soc Mass Spectrom 2005, 16, 324 -332)

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Electrospray ionization mass spectrometry analysis revealed a ∼310 kDa noncovalent hexamer of HPr kinase/phosphatase from Bacillus subtilis

Cited by 17 publications

References 49 publications

Estrogen receptor–ligand complexes measured by chip‐based nanoelectrospray mass spectrometry: An approach for the screening of endocrine disruptors

Estrogen receptor–ligand complexes measured by chip‐based nanoelectrospray mass spectrometry: An approach for the screening of endocrine disruptors

The Role of Initial Oligomers in Amyloid Fibril Formation by Human Stefin B

Structural characterization of sialic acid synthase by electrospray mass spectrometry—A tetrameric enzyme composed of dimeric dimers

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