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61.
Small peptides in serum are potential biomarkers for the diagnosis of cancer and other diseases. The identification of peptide biomarkers in human plasma/serum has become an area of high interest in medical research. However, the direct analysis of peptides in serum samples using mass spectrometry is challenging due to the low concentration of peptides and the high abundance of high-molecular-weight proteins in serum, the latter of which causes severe signal suppression. Herein, we reported that porous semiconductor-noble metal hybrid nanostructures can both eliminate the interference from large proteins in serum samples and significantly enhance the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) yields of peptides captured on the nanostructure. Serum peptide fingerprints with high fidelity can be acquired rapidly, and successful discrimination of colorectal cancer patients based on peptide fingerprints is demonstrated. 相似文献
62.
Shah Hussain Cornelia Pezzei Yüksel Güzel Matthias Rainer Christian W. Huck Günther K. Bonn 《Analytica chimica acta》2014
An efficient blood plasma clean-up method was developed, where methanol protein precipitation was applied, followed by zirconium silicate assisted exclusion of residual proteins. A strong binding of zirconium (IV) silicate to the proteins enabled the elimination of remaining proteins after solvent deproteinization through a rapid solid-phase extraction (SPE) procedure. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF MS) was used for monitoring the proteins during clean-up practice applied to human plasma samples. The proteins were quantified by colorimetric detection using the bicinchoninic acid (BCA) assay. The presented analytical strategy resulted in the depletion of >99.6% proteins from human plasma samples. Furthermore, high-performance liquid chromatography hyphenated to diode-array and electrospray ionization mass spectrometric detection (HPLC–DAD/ESI MS) was applied for qualitative and quantitative analysis of the caffeoylquinic acids (CQAs) and their metabolites in human plasma. The procedure demonstrated high recoveries for the standard compounds spiked at different concentrations. Cynarin and chlorogenic acid were recovered in the range of 81–86% and 78–83%, respectively. Caffeic acid was extracted in the excess of 89–92%, while ferulic acid and dihydroxyhydrocinnamic acid showed a recovery of 87–91% and 92–95%, respectively. The method was partially validated in accordance with FDA-Industry Guidelines for Bioanalytical Method Validation (2001). The presented scheme improves the clean-up efficacy of the methanol deproteinization, significantly reduces the matrix effects and provides a great analytical tool for the isolation of small molecules from human plasma. 相似文献
63.
The interaction of bovine serum albumin (BSA) with the ionic surfactants sodium dodecylsulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) was studied by electron paramagnetic resonance (EPR) spectroscopy of spin label covalently bound to the single free thiol group of the protein. EPR spectra simulation allows to monitor the protein dynamics at the labeling site and to estimate the changes in standard Gibbs free energy, enthalpy and entropy for transferring the nitroxide side chain from the more motionally restricted to the less restricted component. Whereas SDS and CTAC showed similar increases in the dynamics of the protein backbone for all measured concentrations, HPS presented a smaller effect at concentrations above 1.5mM. At 10mM of surfactants and 0.15 mM BSA, the standard Gibbs free energy change was consistent with protein backbone conformations more expanded and exposed to the solvent as compared to the native protein, but with a less pronounced effect for HPS. In the presence of the surfactants, the enthalpy change, related to the energy required to dissociate the nitroxide side chain from the protein, was greater, suggesting a lower water activity. The nitroxide side chain also detected a higher viscosity environment in the vicinity of the paramagnetic probe induced by the addition of the surfactants. The results suggest that the surfactant-BSA interaction, at higher surfactant concentration, is affected by the affinities of the surfactant to its own micelles and micelle-like aggregates. Complementary DLS data suggests that the temperature induced changes monitored by the nitroxide probe reflects local changes in the vicinity of the single thiol group of Cys-34 BSA residue. 相似文献
64.
Pastor E Matveeva E Valle-Gallego A Goycoolea FM Garcia-Fuentes M 《Colloids and surfaces. B, Biointerfaces》2011,88(2):601-609
Mesoporous silicon is a biocompatible, biodegradable material that is receiving increased attention for pharmaceutical applications due to its extensive specific surface. This feature enables to load a variety of drugs in mesoporous silicon devices by simple adsorption-based procedures. In this work, we have addressed the fabrication and characterization of two new mesoporous silicon devices prepared by electrochemistry and intended for protein delivery, namely: (i) mesoporous silicon microparticles and (ii) chitosan-coated mesoporous silicon microparticles. Both carriers were investigated for their capacity to load a therapeutic protein (insulin) and a model antigen (bovine serum albumin) by adsorption. Our results show that mesoporous silicon microparticles prepared by electrochemical methods present moderate affinity for insulin and high affinity for albumin. However, mesoporous silicon presents an extensive capacity to load both proteins, leading to systems were protein could represent the major mass fraction of the formulation. The possibility to form a chitosan coating on the microparticles surface was confirmed both qualitatively by atomic force microscopy and quantitatively by a colorimetric method. Mesoporous silicon microparticles with mean pore size of 35 nm released the loaded insulin quickly, but not instantaneously. This profile could be slowed to a certain extent by the chitosan coating modification. With their high protein loading, their capacity to provide a controlled release of insulin over a period of 60-90 min, and the potential mucoadhesive effect of the chitosan coating, these composite devices comprise several features that render them interesting candidates as transmucosal protein delivery systems. 相似文献
65.
Trenbolone (TRE) is a steroid used by veterinarians on livestock to increase appetite and body weight. The use of TRE has been restricted because of its harmful side effect for consumers. To effectively control TRE residue in food and food product, a rapid and convenient immunoassay was developed by preparing an anti-TRE monoclonal antibody. The immunogen and coating antigen were prepared by coupling TRE hapten with carrier proteins via 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC) method. The optimized method gave an average IC50 value of 0.323 ng mL−1 towards TRE and an average detection limit (LOD) of 0.06 ng mL−1, which is much lower than the maximum residue levels (2.0 ng g−1) accepted by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). The specificity of the antibody was evaluated by measuring cross-reactivity of six structurally related compounds, including 19-nortestosterone (9.7%), testosterone (0.13%), methyltestosterone (<0.01%), methandrostenolone (<0.01%), (+)-dehydroisoandrosterone (<0.001%) and β-estradiol (<0.001%). The recovery rates of the test in detection of TRE-fortified animal tissue, urine and animal feed samples were in the range of 81.3-89.4%, while the intra- and inter-assay coefficients of variation were less than 12.0%. 相似文献
66.
Automated flow-based anion-exchange method for high-throughput isolation and real-time monitoring of RuBisCO in plant extracts 总被引:1,自引:0,他引:1
In this work, a miniaturized, completely enclosed multisyringe-flow system is proposed for high-throughput purification of RuBisCO from Triticum aestivum extracts. The automated method capitalizes on the uptake of the target protein at 4 °C onto Q-Sepharose Fast Flow strong anion-exchanger packed in a cylindrical microcolumn (105 × 4 mm) followed by a stepwise ionic-strength gradient elution (0-0.8 mol/L NaCl) to eliminate concomitant extract components and retrieve highly purified RuBisCO. The manifold is furnished downstream with a flow-through diode-array UV/vis spectrophotometer for real-time monitoring of the column effluent at the protein-specific wavelength of 280 nm to detect the elution of RuBisCO. Quantitation of RuBisCO and total soluble proteins in the eluate fractions were undertaken using polyacrylamide gel electrophoresis (PAGE) and the spectrophotometric Bradford assay, respectively. A comprehensive investigation of the effect of distinct concentration gradients on the isolation of RuBisCO and experimental conditions (namely, type of resin, column dimensions and mobile-phase flow rate) upon column capacity and analyte breakthrough was effected. The assembled set-up was aimed to critically ascertain the efficiency of preliminary batchwise pre-treatments of crude plant extracts (viz., polyethylenglycol (PEG) precipitation, ammonium sulphate precipitation and sucrose gradient centrifugation) in terms of RuBisCO purification and absolute recovery prior to automated anion-exchange column separation. Under the optimum physical and chemical conditions, the flow-through column system is able to admit crude plant extracts and gives rise to RuBisCO purification yields better than 75%, which might be increased up to 96 ± 9% with a prior PEG fractionation followed by sucrose gradient step. 相似文献
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70.
Tania Chowdhury Kaushik Bera Debabrata Samanta Sandip Dolui Suvendu Maity Nakul C. Maiti Prasanta Kumar Ghosh Debasis Das 《应用有机金属化学》2020,34(4):e5556
Four new zinc (II) complexes [Zn (HL1H)Br2] (1), [Zn (HL1H)Cl2] (2), [Zn2(HL2)Br3] (3), and [Zn (HL2)Cl] (4) have been synthesized by adopting template synthetic strategy and utilizing two homologous Schiff base ligands (H2L1 = 4-bromo-2-{[2-(2-hydroxyethylamino)-ethylimino]-methyl}-6-methoxyphenol, H2L2 = 4-bromo-2-{[3-(2-hydroxyethylamino)propylimino]methyl}-6-methoxyphenol), differing in one -CH2- unit in the ligating backbone, by adopting template synthetic strategy. All the complexes have been characterized by single crystal X-ray diffraction analysis as well as by other routine physicochemical techniques. Ligand mediated structural variations have been observed and rationalized by density functional theoretical (DFT) calculations. Interaction of the complexes 1–4 with Bovine Serum Albumin protein (BSA) has been studied by different spectroscopic techniques. A complete thermodynamic profile (ΔHo, ΔSo and ΔGo) was evaluated initially from the change in absorption and fluorescence spectra upon addition of BSA to the complexes. Appreciable binding constant values in the range ~ 0.94–4.51 × 104 M−1 indicate efficient binding tendency of the complexes to BSA with the sequence 1 ≅ 2 > 3 ≅ 4. Circular dichroism (CD), isothermal calorimetric titration experiments, molecular docking and molecular dynamics have been performed to gain deep insight into the binding regions of complex 1 to BSA. Experimental evidences suggest an interaction of zinc complexes at the surface of BSA protein and this particular binding has been exploited to determine unknown concentration of BSA protein. For this purpose complex 1 was explored as a BSA protein quantification tool. 相似文献