Structural insights into calmodulin/adenylyl cyclase 8 interaction

Calmodulin (CaM) is a highly conserved intracellular Ca²⁺-binding protein that exerts important functions in many cellular processes. Prominent examples of CaM-regulated proteins are adenylyl cyclases (ACs), which synthesize cAMP as a central second messenger. The interaction of ACs with CaM represents the link between Ca²⁺-signaling and cAMP-signaling pathways. Thereby, different AC isoforms stimulated by CaM, comprise diverse mechanisms of regulation by the Ca²⁺ sensor. To extend the structural information about the detailed mechanisms underlying the regulation of AC8 by CaM, we employed an integrated approach combining chemical cross-linking and mass spectrometry with two peptides representing the CaM-binding regions of AC8. These experiments reveal that the structures of CaM/AC8 peptide complexes are similar to that of the CaM/skeletal muscle myosin light chain kinase peptide complex where CaM is collapsed around the target peptide that binds to CaM in an antiparallel orientation. Cross-linking experiments were complemented by investigating the binding of AC8 peptides to CaM thermodynamically with isothermal titration calorimetry. There were no hints on a complex, in which both AC8 peptides bind simultaneously to CaM, refining our current understanding of the interaction between CaM and AC8.

Analysis of intercellular communication by flexible hydrodynamic gating on a microfluidic chip

Intercellular Ca²⁺ waves are propagation of Ca²⁺ transients among cells that could be initiated by chemical stimulation. Current methods for analyzing intercellular Ca²⁺ waves are difficult to realize localized chemical stimulations upon the target cell without interfering with adjacent contacting cells. In this paper, a simple and flexible microfluidic method was developed for investigating the intercellular communication of Ca²⁺ signals. A cross-patterned microfluidic chip was designed and fabricated with polydimethylsiloxane as the structural material. Localized chemical stimulation was achieved by a new strategy based on hydrodynamic gating technique. Clusters of target cells were seeded at the location within 300 μm downstream of the intersection of the cross-shaped microchannel. Confined lateral molecular diffusion largely minimized the interference from diffusion-induced stimulation of adjacent cells. Localized stimulation of the target cell with adenosine 5′-triphosphate successfully induced the propagation of intercellular Ca²⁺ waves among a population of adjacent contacting cells. Further inhibition studies verified that the propagation of calcium signals among NIH-3 T3 cells was dependent on direct cytosolic transfer via gap junctions. The developed microfluidic method provides a versatile platform for investigating the dynamics of intercellular communications.

Electron Capture Dissociation of Divalent Metal-adducted Sulfated N-Glycans Released from Bovine Thyroid Stimulating Hormone

Sulfated N-glycans released from bovine thyroid stimulating hormone (bTSH) were ionized with the divalent metal cations Ca²⁺, Mg²⁺, and Co by electrospray ionization (ESI). These metal-adducted species were subjected to infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) and the corresponding fragmentation patterns were compared. IRMPD generated extensive glycosidic and cross-ring cleavages, but most product ions suffered from sulfonate loss. Internal fragments were also observed, which complicated the spectra. ECD provided complementary structural information compared with IRMPD, and all observed product ions retained the sulfonate group, allowing sulfonate localization. To our knowledge, this work represents the first application of ECD towards metal-adducted sulfated N-glycans released from a glycoprotein. Due to the ability of IRMPD and ECD to provide complementary structural information, the combination of the two strategies is a promising and valuable tool for glycan structural characterization. The influence of different metal ions was also examined. Calcium adducts appeared to be the most promising species because of high sensitivity and ability to provide extensive structural information.

Time-resolved fluorescence microscopy for quantitative Ca2+ imaging in living cells

Calcium (Ca²⁺) is a ubiquitous intracellular second messenger and involved in a plethora of cellular processes. Thus, quantification of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and of its dynamics is required for a comprehensive understanding of physiological processes and potential dysfunctions. A powerful approach for studying [Ca²⁺]_i is the use of fluorescent Ca²⁺ indicators. In addition to the fluorescence intensity as a common recording parameter, the fluorescence lifetime imaging microscopy (FLIM) technique provides access to the fluorescence decay time of the indicator dye. The nanosecond lifetime is mostly independent of variations in dye concentration, allowing more reliable quantification of ion concentrations in biological preparations. In this study, the feasibility of the fluorescent Ca²⁺ indicator Oregon Green Bapta-1 (OGB-1) for two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was evaluated. In aqueous solution, OGB-1 displayed a Ca²⁺-dependent biexponential fluorescence decay behaviour, indicating the presence of a Ca²⁺-free and Ca²⁺-bound dye form. After sufficient dye loading into living cells, an in situ calibration procedure has also unravelled the Ca²⁺-free and Ca²⁺-bound dye forms from a global biexponential fluorescence decay analysis, although the dye's Ca²⁺ sensitivity is reduced. Nevertheless, quantitative [Ca²⁺]_i recordings and its stimulus-induced changes in salivary gland cells could be performed successfully. These results suggest that OGB-1 is suitable for 2P-FLIM measurements, which can gain access to cellular physiology.

Ultrasensitive,Rapid, and Selective Detection of Mercury Using Graphene Assisted Laser Desorption/Ionization Mass Spectrometry

We report an extremely sensitive and specific detection of mercuric ions (Hg²⁺) based on graphene assisted laser desorption/ionization mass spectrometry (GALDI-MS). Combining the highly selective coordination interactions between thymine (T) and Hg²⁺, we present a simple, effective, and novel approach, based on π–π interactions of the T-Hg²⁺-T complex and G that can serve as a platform and matrix for GALDI-MS. The present sensor not only exhibits high selectivity and sensitivity (picomolar) to Hg²⁺ in aqueous solution, but also can elucidate the chemical structures of the metal complexes. The significant advantage in the current approach is that there is no need for a sophisticated instrument, and no sample pretreatment is required to detect the Hg²⁺ ions.

Biological and chemical investigation of Allium cepa L. response to selenium inorganic compounds

The aim of this study was to evaluate the biological and chemical response of Allium cepa L. exposed to inorganic selenium compounds. Besides the investigation of the total content of selenium as well as its chemical speciation, the Allium test was used to evaluate the growth of onion roots and mitotic activity in the roots’ meristem. The total content of selenium was determined by inductively coupled plasma mass spectrometry (ICP MS). High-performance liquid chromatography (HPLC), coupled to ICP MS, was used for the selenium chemical speciation. Results indicated that A. cepa plants are able to biotransform inorganic selenium compounds into their organic derivatives, e.g., Se-methylselenocysteine from the Se(IV) inorganic precursor. Although the differences in the biotransformation of selenium are due mainly to the oxidation state of selenium, the experiment has also shown a fine effect of counter ions (H⁺, Na⁺, NH₄ ⁺) on the response of plants and on the specific metabolism of selenium.

Experimental and theoretical study on the complexation of Ca2+ with beauvericin

From extraction experiments and γ-activity measurements, the exchange extraction constant corresponding to the equilibrium Ca²⁺(aq) + 1·Sr²⁺(nb) ? 1·Ca²⁺(nb) + Sr²⁺(aq) taking place in the two-phase water–nitrobenzene system (1 = beauvericin; aq = aqueous phase, nb = nitrobenzene phase) was evaluated as log K _ex(Ca²⁺, 1·Sr²⁺) = 1.1 ± 0.1. Further, the stability constant of the 1·Ca²⁺ complex in nitrobenzene saturated with water was calculated for a temperature of 25 °C: log β _nb(1·Ca²⁺) = 10.1 ± 0.2. Finally, by using quantum mechanical density functional level of theory calculations, the most probable structures of the non-hydrated 1·Ca²⁺ and hydrated 1·Ca²⁺·H₂O complex species were predicted.     

Synergistic extraction of some divalent metal cations into nitrobenzene by using strontium dicarbollylcobaltate and electroneutral macrocyclic lactam receptor

From extraction experiments and $ \gamma $ -activity measurements, the exchange extraction constants corresponding to the general equilibrium M²⁺(aq) + 1·Sr²⁺(nb) $ \Leftrightarrow $ 1·M²⁺(nb) + Sr²⁺(aq) taking place in the two-phase water–nitrobenzene system (M²⁺ = Mg²⁺, Ca²⁺, Ba²⁺, Pb²⁺, Cu²⁺, Zn²⁺, Cd²⁺, $ {\hbox{UO}}_{2}^{2 + } $ , Mn²⁺, Co²⁺, Ni²⁺; 1 = macrocyclic lactam receptor–see Scheme 1; aq = aqueous phase, nb = nitrobenzene phase) were evaluated. Moreover, the stability constants of the 1·M²⁺ complexes in nitrobenzene saturated with water were calculated; they were found to increase in the following cation order: Mg²⁺ < Co²⁺ < Cu²⁺, Mn²⁺, Ni²⁺ < Cd²⁺ < Ca²⁺ < Ba²⁺, Zn²⁺ < Pb²⁺ <  $ {\hbox{UO}}_{2}^{2 + } $ .

Soft Ionization of Saturated Hydrocarbons, Alcohols and Nonpolar Compounds by Negative-Ion Direct Analysis in Real-Time Mass Spectrometry

Large polarizable n-alkanes (approximately C18 and larger), alcohols, and other nonpolar compounds can be detected as negative ions when sample solutions are injected directly into the sampling orifice of the atmospheric pressure interface of the time-of-flight mass spectrometer with the direct analysis in real time (DART) ion source operating in negative-ion mode. The mass spectra are dominated by peaks corresponding to [M + O₂]￣^?. No fragmentation is observed, making this a very soft ionization technique for samples that are otherwise difficult to analyze by DART. Detection limits for cholesterol were determined to be in the low nanogram range.

Multidimensional nano-HPLC coupled with tandem mass spectrometry for analyzing biotinylated proteins

Multidimensional high-performance liquid chromatography (HPLC) is a key method in shotgun proteomics approaches for analyzing highly complex protein mixtures by complementary chromatographic separation principles. Here, we describe an integrated 3D-nano-HPLC/nano-electrospray ionization quadrupole time-of-flight mass spectrometry system that allows an enzymatic digestion of proteins followed by an enrichment and subsequent separation of the created peptide mixtures. The online 3D-nano-HPLC system is composed of a monolithic trypsin reactor in the first dimension, a monolithic affinity column with immobilized monomeric avidin in the second dimension, and a reversed phase C18 HPLC-Chip in the third dimension that is coupled to a nano-ESI-Q-TOF mass spectrometer. The 3D-LC/MS setup is exemplified for the identification of biotinylated proteins from a simple protein mixture. Additionally, we describe an online 2D-nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS setup for the enrichment, separation, and identification of cross-linked, biotinylated species from chemical cross-linking of cytochrome c and a calmodulin/peptide complex using a novel trifunctional cross-linker with two amine-reactive groups and a biotin label.

Probing Protein 3D Structures and Conformational Changes Using Electrochemistry-Assisted Isotope Labeling Cross-Linking Mass Spectrometry

This study presents a new chemical cross-linking mass spectrometry (MS) method in combination with electrochemistry and isotope labeling strategy for probing both protein three-dimensional (3D) structures and conformational changes. For the former purpose, the target protein/protein complex is cross-linked with equal mole of premixed light and heavy isotope labeled cross-linkers carrying electrochemically reducible disulfide bonds (i.e., DSP-d₀ and DSP-d₈ in this study, DSP = dithiobis[succinimidyl propionate]), digested and then electrochemically reduced followed with online MS analysis. Cross-links can be quickly identified because of their reduced intensities upon electrolysis and the presence of doublet isotopic peak characteristics. In addition, electroreduction converts cross-links into linear peptides, facilitating MS/MS analysis to gain increased information about their sequences and modification sites. For the latter purpose of probing protein conformational changes, an altered procedure is adopted, in which the protein in two different conformations is cross-linked using DSP-d₀ and DSP-d₈ separately, and then the two protein samples are mixed in 1:1 molar ratio. The merged sample is subjected to digestion and electrochemical mass spectrometric analysis. In such a comparative cross-linking experiment, cross-links could still be rapidly recognized based on their responses to electrolysis. More importantly, the ion intensity ratios of light and heavy isotope labeled cross-links reveal the conformational changes of the protein, as exemplified by examining the effect of Ca²⁺ on calmodulin conformation alternation. This new cross-linking MS method is fast and would have high value in structural biology.

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Graphical Abstract ?

     

Why do the Abundances of Ions Generated by MALDI Look Thermally Determined?

In a previous study (J. Mass Spectrom. 48, 299–305, 2013), we observed that the abundance of each ion in a matrix-assisted laser desorption ionization (MALDI) spectrum looked thermally determined. To find out the explanation for the phenomenon, we estimated the ionization efficiency and the reaction quotient (Q_A) for the autoprotolysis of matrix, M + M → [M + H]⁺ + [M ? H]^?, from the temperature-controlled laser desorption ionization spectra of α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). We also evaluated the equilibrium constants (K_A) for the autoprotolysis at various temperatures by quantum chemical calculation. Primary ion formation via various thermal models followed by autoprotolysis-recombination was compatible with the observations. The upper limit of the effective temperature of the plume where autoprotolysis-recombination occurs was estimated by equating Q_A with the calculated equilibrium constant.

Analysis of a Soluble (UreD:UreF:UreG)2 Accessory Protein Complex and Its Interactions with Klebsiella aerogenes Urease by Mass Spectrometry

Maturation of the nickel-containing urease of Klebsiella aerogenes is facilitated by the UreD, UreF, and UreG accessory proteins along with the UreE metallo-chaperone. A fusion of the maltose binding protein and UreD (MBP-UreD) was co-isolated with UreF and UreG in a soluble complex possessing a (MBP-UreD:UreF:UreG)₂ quaternary structure. Within this complex a UreF:UreF interaction was identified by chemical cross-linking of the amino termini of its two UreF protomers, as shown by mass spectrometry of tryptic peptides. A pre-activation complex was formed by the interaction of (MBP-UreD:UreF:UreG)₂ and urease. Mass spectrometry of intact protein species revealed a pathway for synthesis of the urease pre-activation complex in which individual hetero-trimer units of the (MBP-UreD:UreF:UreG)₂ complex bind to urease. Together, these data provide important new insights into the structures of protein complexes associated with urease activation.

Improved DFT-Based Interpretation of ESI-MS of Aqueous Metal Cations

We present results showing that our recently developed density functional theory (DFT)-based speciation model of the aqueous Al³⁺ system has the potential to improve the interpretations of ESI-MS studies of aqueous metal cation hydrolytic speciation. The main advantages of our method are that (1) it allows for the calculation of the relative abundance of a given species which may be directly assigned to the signal intensity in a mass spectrum; (2) in cases where species with identical m?z ratios may coexist, the assignment can be unambiguously assigned based on their theoretical relative abundances. As a demonstration of its application, we study four pairs of monomer and dimer aqueous Al³⁺ species, each with identical m/z ratio. For some of these pairs our method predicts that the dominant species changes from the monomer to the dimer species under varying pH conditions.

Elimination of Benzene from Protonated N-Benzylindoline: Benzyl Cation/Proton Transfer or Direct Proton Transfer?

Collision-induced dissociation (CID) of protonated N-benzylindoline and its derivatives was investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Elimination of benzene was observed besides hydride transfer and electron transfer reactions. D-labeling experiments and accurate mass determinations of the product ions confirm that the external proton is retained in the fragment ion, and the elimination reaction was proposed to be initiated by benzyl cation transfer rather than proton transfer. Benzyl cation transfer from the nitrogen atom to one of the sp²-hybridized carbon atoms in the indoline core is the key step, and subsequent proton transfer reaction leads to the elimination of benzene. Density functional theory (DFT)-based calculations were performed and the computational results also support the benzyl cation/proton transfer mechanism.

Plasma-Assisted Reaction Chemical Ionization for Elemental Mass Spectrometry of Organohalogens

We present plasma-assisted reaction chemical ionization (PARCI) for elemental analysis of halogens in organic compounds. Organohalogens are broken down to simple halogen-containing molecules (e.g., HBr) in a helium microwave-induced plasma followed by negative mode chemical ionization (CI) in the afterglow region. The reagent ions for CI originate from penning ionization of gases (e.g., N₂) introduced into the afterglow region. The performance of PARCI-mass spectrometry (MS) is evaluated using flow injection analyses of organobromines, demonstrating 5–8 times better sensitivities compared with inductively coupled plasma MS. We show that compound-dependent sensitivities in PARCI-MS mainly arise from sample introduction biases.

Application of UPLC-QTOF-MS in MSE mode for the rapid and precise identification of alkaloids in goldenseal (Hydrastis canadensis)

Here, we describe a new application of ultra-performance liquid chromatography coupled with an electrospray ionization quadrupole time-of-flight mass spectrometry operating in MS^E mode (UPLC-QTOF-MS^E) for the sensitive, fast, and effective characterization of alkaloids in goldenseal (Hydrastis canadensis). This approach allowed identification of alkaloids using a cyclic low and high collision energy spectral acquisition mode providing simultaneous accurate precursor and fragment ion mass information. A total of 45 compounds were separated and 40 of them characterized including one new compound and 7 identified for the first time in goldenseal. The spectral data obtained using this method is comparable to those obtained by conventional LC-MSⁿ. However, the UPLC-QTOF-MS^E method offers high chromatographic resolution with structural characterization facilitated by accurate mass measurement in both MS and MS/MS modes in a single analytical run; this makes it suitable for the rapid analysis and screening of alkaloids in plant extracts.

New garnet compounds A 3 2+ B 2 2+ C4+V 2 5+ O12(A = Ca,Cd; B = Mg,Zn, Co,Ni, Cu,Mn, Cd; C = Ge,Si)

Garnet compounds A ₃ ²⁺ B ₂ ²⁺ C⁴⁺V ₂ ⁵⁺ O₁₂ (A = Ca, Cd; B = Mg, Zn, Co, Ni, Cu, Mn, Cd; C = Ge, Si) (space group \(Ia\bar 3d\) , Z = 8) have been prepared by solid-phase synthesis in air at 900–1250°C. Most of these compounds melt incongruently or decompose in the solid phase. The isomorphic capacity of garnets and their homogeneity fields are discussed. The structures of Ca₃Zn₂GeV₂O₁₂ and Ca₃Cu₂GeV₂O₁₂ have been refined by the Rietveld method.     

Preconcentration of trace aluminum (III) ion using a nanometer-sized TiO2-silica composite modified with 4-aminophenylarsonic acid, and its determination by ICP-OES

We describe a nanometer sized composite material made from titanium dioxide and silica that was chemically modified with 4-aminophenylarsonic acid and used for selective solid-phase extraction, separation and preconcentration of of aluminum(III) prior to its determination by ICP-OES. Under optimized conditions, the static adsorption capacity is 56.58?mg?g^?1, the enrichment factor is 150, the relative standard deviation is 1.6% (for n?=?11), and the detection limit (3?s) is 60?pg?mL^?1. The method was validated by analyzing the reference materials GBW 09101 (hair) and GBW 10024 (scallop) and successfully applied to the determination of trace Al(III) in spiked water samples and human urine, with recoveries ranging from 96% to 101%.