Organelle sensor: amperometric determination of nadh by immobilized mitochondrial electron transport particles

A new sensor for NADH was developed by making use of an immobilized subcellular organelle. Mitochondria was used as a model system for assembling an organelle sensor. Mitochondrial electron transport particles (ETP) were prepared from beef heart muscle and entrapped in the membrane formed of agar gel. The membrane-bound ETP was found capable of NADH oxidation: $$NADH + \tfrac{1}{2}O_2 + H^ + \xrightarrow{{ETP}}NAD^ - + H_2 O$$ The membrane was tightly attached to the surface of an oxygen electrode capable of amperometric detection of O2. The sensor responded to NADH in solution with a resulting electric output. The response was enhanced by the addition of 2,4-dinitrophenol (DNP). NADH was determined in the concentration range 1–300 µM. NADH was alternatively determined for 2 weeks without replacing the ETP-bound membrane.     

Recycling of NADP+ using immobilizedE. coli and glucose-6-phosphate dehydrogenase

Recycling of NADP⁺ using immobilized wholeEscherichia coli cells as source of respiratory chain, glucose-6-phosphate, and soluble yeast glucose-6-phosphate dehydrogenase (1.1.1.49) is described. NADP⁺ was recycled more than 10-fold. We demonstrated NADPH respiration at pH 5.8 inE. coli membrane vesicles. The respiratory chain was involved most probably in NADPH oxidation.

1. The respiratory activity is localized at the level of the inner bacterial membrane. The active site for NADPH facing the cytoplasm.
2. NADPH respiration is inhibited by 10 mM cyanide, similar to the conditions of inhibition of NADH respiration.
3. NADPH dehydrogenase activity seems to be the limiting step of the respiratory chain:K _M for NADPH respiration and NADPH dehydrogenase activity are similar. The pH optima for these two activities are also comparable (around pH 5.8). Furthermore, the following properties are rather in favor of a common NADH dehydrogenase and NADPH dehydrogenase activity (1.6.99.2).

o| li](1)|At saturating concentrations of NADH and NADPH, neither respiration nor dehydrogenase activities were additive. li](2)|Similar heat inactivation kinetics were observed for NADH and NADPH dehydrogenase-activity. Protection against heat inactivation was obtained for the two activities with NAD⁺, NADP⁺, NADH, and NADPH. All these results suggested the possibility of recycling of NADP⁺ under similar conditions to those previously described for NAD⁺ (Burstein et al., 1981). It becomes thus possible to use various NAD⁺ and NADP⁺-dependent dehydrogenases in enzyme technology.     

Top-Down and Bottom-Up Identification of Proteins by Liquid Extraction Surface Analysis Mass Spectrometry of Healthy and Diseased Human Liver Tissue

Liquid extraction surface analysis mass spectrometry (LESA MS) has the potential to become a useful tool in the spatially-resolved profiling of proteins in substrates. Here, the approach has been applied to the analysis of thin tissue sections from human liver. The aim was to determine whether LESA MS was a suitable approach for the detection of protein biomarkers of nonalcoholic liver disease (nonalcoholic steatohepatitis, NASH), with a view to the eventual development of LESA MS for imaging NASH pathology. Two approaches were considered. In the first, endogenous proteins were extracted from liver tissue sections by LESA, subjected to automated trypsin digestion, and the resulting peptide mixture was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (bottom-up approach). In the second (top-down approach), endogenous proteins were extracted by LESA, and analyzed intact. Selected protein ions were subjected to collision-induced dissociation (CID) and/or electron transfer dissociation (ETD) mass spectrometry. The bottom-up approach resulted in the identification of over 500 proteins; however identification of key protein biomarkers, liver fatty acid binding protein (FABP1), and its variant (Thr→Ala, position 94), was unreliable and irreproducible. Top-down LESA MS analysis of healthy and diseased liver tissue revealed peaks corresponding to multiple (~15–25) proteins. MS/MS of four of these proteins identified them as FABP1, its variant, α-hemoglobin, and 10 kDa heat shock protein. The reliable identification of FABP1 and its variant by top-down LESA MS suggests that the approach may be suitable for imaging NASH pathology in sections from liver biopsies. Graphical Abstract

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Acetonitrile Adduct Formation as a Sensitive Means for Simple Alcohol Detection by LC-MS

Simple alcohols formed protonated acetonitrile adducts containing up to two acetonitrile molecules when analyzed by ESI or APCI in the presence of acetonitrile in the solvent. These acetonitrile adducts underwent dissociation to form a nitrilium ion, also referred to as the substitution ion. Diols and triols behaved differently. In ESI, they formed only one acetonitrile adduct containing one acetonitrile. The S ion was not observed in ESI and was only weakly observed from the dissociation of the (M?+?ACN?+?H)⁺ ion. On the other hand, the S ion was abundantly formed from the diols in APCI. This formation of acetonitrile adducts and substitution ion from simple alcohols/diols offers an opportunity to detect simple alcohols/diols sensitively by LC-MS interfaced by ESI or APCI. The utility of this chemistry was demonstrated in a method developed for the quantification of cyclohexanol in rat plasma by monitoring the CID-induced fragmentation from the S ion to a fragment ion. Graphical Abstract

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Synthesis of the bifuncttonal dinucleotide AMP-ATP and its application in general ligand affinity chromatography

A new AMP derivative substituted with spacer arms both at position N⁶ and C8 of the adenine moiety was synthesized and immobilized to Sepharose. To the immobilized ligand was subsequently coupled C8-substituted ATP in a solid-phase synthesis fashion yielding the bifunctional general ligand AMP-ATP. This affinity material was used in the separation of two major groups of enzymes, dehydrogenases and kinases. It was found that on passage of crude homogenates obtained from mouse kidney through the affinity column, several dehydrogenases and kinases were bound, which could be eluted separately using pulses of NADH and ATP, respectively. In the fractions obtained on NADH elution, lactate dehydrogenase, malate dehydrogenase, and α-glycerol phosphate dehydrogenase were found, whereas ATP eluted 3-phosphoglyceric acid kinase, pyruvate kinase, and aldolase.     

Investigation of bn-44 Peptide Fragments Using High Resolution Mass Spectrometry and Isotope Labeling

An N-terminal deuterohemin-containing hexapeptide (DhHP-6) was designed as a short peptide cytochrome c (Cyt c) mimetic to study the effect of N-terminal charge on peptide fragmentation pathways. This peptide gave different dissociation patterns than normal tryptic peptides. Upon collision-induced dissociation (CID) with an ion trap mass spectrometer, the singly charged peptide ion containing no added proton generated abundant and characteristic b_n-44 ions instead of b_n-28 (a_n) ions. Studies by high resolution mass spectrometry (HRMS) and isotope labeling indicate that elimination of 44 Da fragments from b ions occurs via two different pathways: (1) loss of CH₃CHO (44.0262) from a Thr side chain; (2) loss of CO₂ (43.9898) from the oxazolone structure in the C-terminus. A series of analogues were designed and analyzed. The experimental results combined with Density Functional Theory (DFT) calculations on the proton affinity of the deuteroporphyrin demonstrate that the production of these novel b_n-44 ions is related to the N-terminal charge via a charge-remote rather than radical-directed fragmentation pathway. Graphical Abstract

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Characterization of poly(2-vinylpyridine)-block-poly(methyl methacrylate) copolymers and blends of their homopolymers by liquid chromatography at critical conditions

Poly(2-vinylpyridine)s (P2VPs) are important polymers with extensive applications in modern day material science. P2VP is an exceptional case for liquid chromatography because of certain polar interactions with most of the stationary phases. In the present study, we established the critical adsorption point (CAP) of P2VP for the first time. The effectiveness of the method is demonstrated by analyses of blends and block copolymers of P2VP and PMMA. The CAP of PMMA is established for determination of molar mass of P2VP component of above mentioned blends and block copolymers. The methods successfully demonstrate the separation of both types of homopolymers from the rest of the samples in conjunction with the determination of molar mass distribution of noncritical block or component. Graphical Abstract

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Initial Velocity Distribution of MALDI/LDI Ions Measured by Internal MALDI Source Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry

A new method for measuring the ion velocity distribution using an internal matrix-assisted laser desorption/ionization (MALDI) source Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer is described. The method provides the possibility of studying ion velocities without any influence of electric fields in the direction of the instrument axis until the ions reach the ICR cell. It also allows to simultaneously account for and to estimate not only the velocity distribution but the angular distribution as well. The method was demonstrated using several types of compounds in laser desorption/ionization (LDI) mode. Graphical Abstract

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Biomolecular Imaging with a C60-SIMS/MALDI Dual Ion Source Hybrid Mass Spectrometer: Instrumentation,Matrix Enhancement,and Single Cell Analysis

We describe a hybrid MALDI/C₆₀-SIMS Q-TOF mass spectrometer and corresponding sample preparation protocols to image intact biomolecules and their fragments in mammalian spinal cord, individual invertebrate neurons, and cultured neuronal networks. A lateral spatial resolution of 10 μm was demonstrated, with further improvement feasible to 1 μm, sufficient to resolve cell outgrowth and interconnections in neuronal networks. The high mass resolution (>13,000 FWHM) and tandem mass spectrometry capability of this hybrid instrument enabled the confident identification of cellular metabolites. Sublimation of a suitable matrix, 2,5-dihydroxybenzoic acid, significantly enhanced the ion signal intensity for intact glycerophospholipid ions from mammalian nervous tissue, facilitating the acquisition of high-quality ion images for low-abundance biomolecules. These results illustrate that the combination of C₆₀-SIMS and MALDI mass spectrometry offers particular benefits for studies that require the imaging of intact biomolecules with high spatial and mass resolution, such as investigations of single cells, subcellular organelles, and communities of cells. Graphical Abstract

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Micellar solubilization of biopolymers in hydrocarbon solvents. ii. the case of horse liver alcohol dehydrogenase

The chemical characterization of horse liver alcohol dehydrogenase solubilized in isooctane via reverse micelles formed by the anionic surfactant di (2-ethyl-hexyl) sodium sulfosuccinate (AOT) and water (0.6 to 4% v/v) is presented. The enzyme’s catalytic activity toward acetaldehyde reduction is markedly dependent upon w0 = [H2O]/[AOT], and upon the pH of the stock aqueous solution (pHst), from which the hydrocarbon enzyme solution is prepared. Kinetically, the micellar solution appears to follow a normal Michaelis-Menten behavior, with a turnover number which, under the optimal conditions (w0 = 42, pHst = 8.8), appears to be higher than in bulk water. The affinity between enzyme and NADH, as judged from direct binding studies (quenching of the protein fluorescence), is much reduced with respect to water if concentrations refer to the water pool of the micelles, and comparable to water if concentrations refer to the overall volume (hydrocarbon plus water pool). Also, the Km values are much higher if concentrations refer to the water pool. Ultraviolet absorption studies show that the aromatic chromophores are not significantly perturbed on going from a water solution to the micellar solution. The essentially aqueous environment of the protein in the reverse micelles is confirmed by fluoresence studies. Circular dichroism studies show that the enzyme’s conformation in the micelles is similar to that in water; however, under certain conditions, small but significant changes of the main chain folding seem to occur, which do not impair enzymatic activity. The spectroscopic properties of NADH in the hydrocarbon phase (fluorescence and circular dichroism) are also investigated. The potential of the LADH-NADH system for technical applications (oxidoreduction of lipophylic substrates) is discussed.     

More Than Charged Base Loss — Revisiting the Fragmentation of Highly Charged Oligonucleotides

Tandem mass spectrometry is a well-established analytical tool for rapid and reliable characterization of oligonucleotides (ONs) and their gas-phase dissociation channels. The fragmentation mechanisms of native and modified nucleic acids upon different mass spectrometric activation techniques have been studied extensively, resulting in a comprehensive catalogue of backbone fragments. In this study, the fragmentation behavior of highly charged oligodeoxynucleotides (ODNs) comprising up to 15 nucleobases was investigated. It was found that ODNs exhibiting a charge level (ratio of the actual to the total possible charge) of 100% follow significantly altered dissociation pathways compared with low or medium charge levels if a terminal pyrimidine base (3' or 5') is present. The corresponding product ion spectra gave evidence for the extensive loss of a cyanate anion (NCO^–), which frequently coincided with the abstraction of water from the 3'- and 5'-end in the presence of a 3'- and 5'-terminal pyrimidine nucleobase, respectively. Subsequent fragmentation of the M-NCO^– ion by MS³ revealed a so far unreported consecutive excision of a metaphosphate (PO₃ ^–)-ion for the investigated sequences. Introduction of a phosphorothioate group allowed pinpointing of PO₃ ^– loss to the ultimate phosphate group. Several dissociation mechanisms for the release of NCO^– and a metaphosphate ion were proposed and the validity of each mechanism was evaluated by the analysis of backbone- or sugar-modified ONs.

Highly selective piezoelectric sensor for lead(II) based on the lead-catalyzed release of gold nanoparticles from a self-assembled nanosurface

A novel quartz crystal microbalance (QCM) sensor has been developed for highly selective and sensitive detection of Pb²⁺ by exploiting the catalytic effect of Pb²⁺ ions on the leaching of gold nanoparticles from the surface of a QCM sensor. The use of self-assembled gold nanoparticles (AuNPs) strongly enlarges the size of the interface and thus amplifies the analytical response resulting from the loss of mass. This results in a very low detection limit for Pb²⁺ (30 nM). The high selectivity is demonstrated by studying the effect of potentially interfering ions both in the absence and presence of Pb²⁺ ions. This simple and well reproducible sensor was applied to the determination of lead in the spiked drinking water. This work provides a novel strategy for fabricating QCM sensors towards Pb²⁺ in real samples. Figure

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ECD of Tyrosine Phosphorylation in a Triple Quadrupole Mass Spectrometer with a Radio-Frequency-Free Electromagnetostatic Cell

A radio frequency-free electromagnetostatic (EMS) cell devised for electron-capture dissociation (ECD) of ions has been retrofitted into the collision-induced dissociation (CID) section of a triple quadrupole mass spectrometer to enable recording of ECD product-ion mass spectra and simultaneous recording of ECD-CID product-ion mass spectra. This modified instrument can be used to produce easily interpretable ECD and ECD-CID product-ion mass spectra of tyrosine-phosphorylated peptides that cover over 50% of their respective amino-acid sequences and readily identify their respective sites of phosphorylation. ECD fragmentation of doubly protonated, tyrosine-phosphorylated peptides, which was difficult to observe with FT-ICR instruments, occurs efficiently in the EMS cell. Figure

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Action of the transferase activity of phospholipase D on rat liver mitochondria

The action of phospholipase D on rat liver mitochondria in the presence of methanol, glycerol, and ethanolamine has been studied. The phospholipid compositions of the modified and native mitochondria have been determined. Incubation of the mitochondria with phospholipase D led to a considerable decrease in the activities of cytochrome c oxidase and NADH-cytochrome reductase.     

In Situ Characterization of Proteins Using Laserspray Ionization on a High-Performance MALDI-LTQ-Orbitrap Mass Spectrometer

The MALDI-LTQ-Orbitrap XL mass spectrometer is a high performance instrument capable of high resolution and accurate mass (HRAM) measurements. The maximum m/z of 4000 precludes the MALDI analysis of proteins without generating multiply charged ions. Herein, we present the study of HRAM laserspray ionization mass spectrometry (MS) with MS/MS and MS imaging capabilities using 2-nitrophloroglucinol (2-NPG) as matrix on a MALDI-LTQ-Orbitrap XL mass spectrometer. The optimized conditions for multiply charged ion production have been determined and applied to tissue profiling and imaging. Biomolecules as large as 15 kDa have been detected with up to five positive charges at 100 K mass resolution (at m/z 400). More importantly, MS/MS and protein identification on multiply charged precursor ions from both standards and tissue samples have been achieved for the first time with an intermediate-pressure source. The initial results reported in this study highlight potential utilities of laserspray ionization MS analysis for simultaneous in situ protein identification, visualization, and characterization from complex tissue samples on a commercially available HRAM MALDI MS system. Graphical Abstract

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d- and l-amino acids in Antarctic lakes: assessment of a very sensitive HPLC-MS method

Amino acids represent a fraction of organic matter in marine and freshwater ecosystems, and a source of carbon, nitrogen and energy. l-Amino acids are the most common enantiomers in nature because these chiral forms are used during the biosynthesis of proteins and peptide. To the contrary, the occurrence of d-amino acids is usually linked to the presence of bacteria. We investigated the distribution of l- and d-amino acids in the lacustrine environment of Terra Nova Bay, Antarctica, in order to define their natural composition in this area and to individuate a possible relationship with primary production. A simultaneous chromatographic separation of 40 l- and d-amino acids was performed using a chiral stationary phase based on teicoplainin aglycone (chirobiotic tag). The chromatographic separation was coupled to two different mass spectrometers—an LTQ-Orbitrap XL (Thermo Fisher Scientific) and an API 4000 (ABSciex)—in order to investigate their quantitative performance. High-performance liquid chromatography coupled with mass spectrometry methods were evaluated through the estimation of their linear ranges, repeatability, accuracy and detection and quantification limits. The high-resolution mass spectrometer LTQ-Orbitrap XL presented detection limits between 0.4 and 7 μg?l ^?1, while the triple quadrupole mass spectrometer API 4000 achieved the best detection limits reported in the literature for the quantification of amino acids (between 4 and 200 ng?l ^?1). The most sensitive method, HPLC-API 4000, was applied to lake water samples. Figure

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Direct electrochemistry of glucose oxidase and sensing glucose using a screen-printed carbon electrode modified with graphite nanosheets and zinc oxide nanoparticles

We have studied the direct electrochemistry of glucose oxidase (GOx) immobilized on electrochemically fabricated graphite nanosheets (GNs) and zinc oxide nanoparticles (ZnO) that were deposited on a screen printed carbon electrode (SPCE). The GNs/ZnO composite was characterized by using scanning electron microscopy and elemental analysis. The GOx immobilized on the modified electrode shows a well-defined redox couple at a formal potential of ?0.4 V. The enhanced direct electrochemistry of GOx (compared to electrodes without ZnO or without GNs) indicates a fast electron transfer at this kind of electrode, with a heterogeneous electron transfer rate constant (K_s) of 3.75 s^?1. The fast electron transfer is attributed to the high conductivity and large edge plane defects of GNs and good conductivity of ZnO-NPs. The modified electrode displays a linear response to glucose in concentrations from 0.3 to 4.5 mM, and the sensitivity is 30.07 μA mM^?1 cm^?2. The sensor exhibits a high selectivity, good repeatability and reproducibility, and long term stability. Figure

Graphical representation for the fabrication of GNs/ZnO composite modified SPCE and the immobilization of GOx     

Characterization of Stress-Exposed Granulocyte Colony Stimulating Factor Using ELISA and Hydrogen/Deuterium Exchange Mass Spectrometry

Information on the higher-order structure is important in the development of biopharmaceutical drugs. Recently, hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) has been widely used as a tool to evaluate protein conformation, and unique automated systems for HDX-MS are now commercially available. To investigate the potential of this technique for the prediction of the activity of biopharmaceuticals, granulocyte colony stimulating factor (G-CSF), which had been subjected to three different stress types, was analyzed using HDX-MS and through comparison with receptor-binding activity. It was found that HDX-MS, in combination with ion mobility separation, was able to identify conformational changes in G-CSF induced by stress, and a good correlation with the receptor-binding activity was demonstrated, which cannot be completely determined by conventional peptide mapping alone. The direct evaluation of biological activity using bioassay is absolutely imperative in biopharmaceutical development, but HDX-MS can provide the alternative information in a short time on the extent and location of the structural damage caused by stresses. Furthermore, the present study suggests the possibility of this system being a versatile evaluation method for the preservation stability of biopharmaceuticals. Graphical Abstract

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Direct Analysis in Real Time-Mass Spectrometry for the Rapid Detection of Metabolites of Aconite Alkaloids in Intestinal Bacteria

In the present work, direct analysis of real time ionization combined with multi-stage tandem mass spectrometry (DART-MSⁿ) was used to investigate the metabolic profile of aconite alkaloids in rat intestinal bacteria. A total of 36 metabolites from three aconite alkaloids were identified by using DART-MSⁿ, and the feasibility of quantitative analysis of these analytes was examined. Key parameters of the DART ion source, such as helium gas temperature and pressure, the source-to-MS distance, and the speed of the autosampler, were optimized to achieve high sensitivity, enhance reproducibility, and reduce the occurrence of fragmentation. The instrument analysis time for one sample can be less than 10 s for this method. Compared with ESI-MS and UPLC-MS, the DART-MS is more efficient for directly detecting metabolic samples, and has the advantage of being a simple, high-speed, high-throughput method. Graphical Abstract

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Determination of Internal Energy Distributions of Laser Electrospray Mass Spectrometry using Thermometer Ions and Other Biomolecules

The internal energy distributions for dried and liquid samples that were vaporized with femtosecond duration laser pulses centered at 800 nm and postionized by electrospray ionization-mass spectrometry (LEMS) were measured and compared with conventional electrospray ionization mass spectrometry (ESI-MS). The internal energies of the mass spectral techniques were determined by plotting the ratio of the intact parent molecular features to all integrated ion intensities of the fragments as a function of collisional energy using benzylpyridinium salts and peptides. Measurements of dried p-substituted benzylpyridinium salts using LEMS resulted in a greater extent of fragmentation in addition to the benzyl cation. The mean relative internal energies, int> were determined to be 1.62?±?0.06, 2.0?±?0.5, and 1.6?±?0.3 eV for ESI-MS, dried LEMS, and liquid LEMS studies, respectively. Two-photon resonances with the laser pulses likely caused lower survival yields in LEMS analyses of dried samples but not liquid samples. In studies with larger biomolecules, LEMS analyses of dried samples from glass showed a decrease in survival yield compared with conventional ESI-MS for leucine enkephalin and bradykinin of ~15% and 11%, respectively. The survival yields for liquid LEMS analyses were comparable to or better than ESI-MS for benzylpyridinium salts and large biomolecules. Figure

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