Evaluation of cyclooxygenase-2 inhibitors using pulsed ultrafiltration mass spectrometry

Since selective inhibition of the inducible form of cyclooxygenase (COX-2) might retain all the benefits of classical nonsteroidal antiinflammatory agents while avoiding the major side effects associated with inhibition of the constitutive isoform COX-1, COX-2 has become an important target for the discovery and development of new antiinflammatory drugs. To aid in the discovery and characterization of such selective inhibitors, we have applied a mass spectrometry-based screening technique, pulsed ultrafiltration mass spectrometry, using COX-2 as the target. In a blind study, 18 samples enriched with one or more inhibitors of COX-2 were evaluated. The matrixes for the test samples consisted of DMSO, r DMSO solutions of a plant extract, or a bacterial fermentation broth extract. The composition of the samples was unknown during the assays, as were the concentrations of the COX-2 inhibitors. A soluble recombinant form of human COX-2 was incubated with each sample, and then an aliquot of each mixture was injected into the stirred ultrafiltration chamber fitted with a 30000 MW cutoff ultrafiltration membrane. After the unbound and weakly bound compounds were washed away, the ligand-receptor complexes were disrupted using an acidified 10% methanol solution. The released ligands were trapped on a C18 cartridge and then identified using liquid chromatography-negative ion electrospray mass spectrometry with the trapping cartridge as the HPLC column. Neither the plant matrix nor the fermentation broth extract were found to interfere with the assay. Two or three ligands for COX-2 were identified in each sample, which included polar and nonpolar compounds and inhibitors with IC50 values ranging from 100 microM to 10 nM.     

Screening of inhibitors using enzymes entrapped in sol-gel-derived materials

In recent years, a number of new methods have been reported that make use of immobilized enzymes either on microarrays or in bioaffinity columns for high-throughput screening of compound libraries. A key question that arises in such methods is whether immobilization may alter the intrinsic catalytic and inhibition constants of the enzyme. Herein, we examine how immobilization within sol-gel-derived materials affects the catalytic constant (kcat), Michaelis constant (KM), and inhibition constant (KI) of the clinically relevant enzymes Factor Xa, dihydrofolate reductase, cyclooxygenase-2, and gamma-glutamyl transpeptidase. These enzymes were encapsulated into sol-gel-derived glasses produced from either tetraethyl orthosilicate (TEOS) or the newly developed silica precursor diglyceryl silane (DGS). It was found that the catalytic efficiency and long-term stability of all enzymes were improved upon entrapment into DGS-derived materials relative to entrapment in TEOS-based glasses, likely owing to the liberation of the biocompatible reagent glycerol from DGS. The KM values of enzymes entrapped in DGS-derived materials were typically higher than those in solution, whereas upon entrapment, kcat values were generally lowered by a factor of 1.5-7 relative to the value in solution, indicating that substrate turnover was limited by partitioning effects or diffusion through the silica matrix. Nonetheless, the apparent KI value for the entrapped enzyme was in most cases within error of the value in solution, and even in the worst case, the values differed by no more than a factor of 3. The implications of these findings for high-throughput screening are discussed.     

Microbial strain prioritization using metabolomics tools for the discovery of natural products

Natural products profoundly impact many research areas, including medicine, organic chemistry, and cell biology. However, discovery of new natural products suffers from a lack of high throughput analytical techniques capable of identifying structural novelty in the face of a high degree of chemical redundancy. Methods to select bacterial strains for drug discovery have historically been based on phenotypic qualities or genetic differences and have not been based on laboratory production of secondary metabolites. Therefore, untargeted LC/MS-based secondary metabolomics was evaluated to rapidly and efficiently analyze marine-derived bacterial natural products using LC/MS-principal component analysis (PCA). A major goal of this work was to demonstrate that LC/MS-PCA was effective for strain prioritization in a drug discovery program. As proof of concept, we evaluated LC/MS-PCA for strain selection to support drug discovery, for the discovery of unique natural products, and for rapid assessment of regulation of natural product production.     

Identification of natural products using HPLC-SPE combined with CapNMR

Two major development areas in HPLC-NMR hyphenation are postcolumn solid-phase extraction (HPLC-SPE-NMR) and capillary separations with NMR detection by means of solenoidal microcoils (CapNMR). These two techniques were combined off-line into HPLC-SPE-CapNMR, which combines the advantage of high loadability of normal-bore HPLC columns with high mass sensitivity of capillary NMR probes with an active volume of 1.5 microL. The technique was used for rapid identification of complex sesquiterpene lactones and esterified phenylpropanoids present in an essentially crude plant extract (toluene fraction of an ethanolic extract of Thapsia garganica fruits). Elution profiles of 10 x 1 mm i.d. SPE cartridges filled with poly(divinylbenzene) resin were found to be only marginally broader than those observed upon direct injection of 6-microL samples into the probe. Thus, the technique focuses analytes emerging in the HPLC elution bands of 0.5-1 mL into volumes of approximately 10 microL, compatible with the CapNMR probe. Using this technique, nine natural products (1-9) present in the plant extract in amounts varying from 0.1 to 20% were identified by means of 1D and 2D NMR spectra, supported by parallel HPLC-ESIMS measurements. Therefore, HPLC-SPE-CapNMR should be regarded as an attractive alternative to other applications of CapNMR for mixture analysis.     

Screening the fabrication conditions of ultrafiltration membranes by using the uniform design and regression analysis methods

In this work, the concerned casting conditions for the fabrication of the ultrafiltration (UF) membrane from polysulfone were the gelation medium used, the temperature of the gelation bath, the gelation period, and the evaporation time of the cast sheet. The uniform design method was applied to substantially reduce the number of experiments for studying the influence of each of four casting conditions introduced in the preparation of UF-membranes. The pure water flux and the molecular weight cutoff (MWCO) of each of the four macromolecule-standards, namely egg white lysozyme, egg white albumin, bovine serum albumin and bovine γ-globulius, of each prepared UF-membrane were determined. These experimental responses were then analyzed by using a non-linear model with the backward regression method. The results of this work showed that the uniform design-non linear model (UD-NLM) method can substantially reduce the number of experimental runs for predicting the influences of the studied casting conditions on the ultrafiltration performance of the prepared asymmetric polysulfone membranes.     

超滤在天然甘露醇精制中的应用

在天然甘露醇的生产中 ,应用中空纤维超滤技术改进传统的精制工艺 ,去除糖胶、颗粒等杂质 .纯化后的产品可改善大输液的生产工艺 ,满足出口产品的要求 .近两年的实际应用表明 ,该新技术的开发和应用是成功的 .     

Screening of glycosylation patterns in serum using natural glycoprotein microarrays and multi-lectin fluorescence detection

Protein glycosylation has been implicated in key biological processes including immunological recognition, cellular adhesion, protein folding, and signaling as well as disease progression. Although several methods are available to assess glycosylation of protein structures, none of them is able to screen complex biological samples at a global as well as an individual scale. A novel strategy presented here uses an all-liquid phase enrichment and prefractionation methodology coupled to glycoprotein microarray technology using a multiple lectin-based, biotin-streptavidin detection scheme. Selective detection of glycan structures was made possible by employing multiple lectins to screen glycoprotein standards as well as serum samples from normal subjects or patients with chronic pancreatitis or pancreatic cancer. Interestingly, in some instances, a greater degree of glycosylation was seen in proteins that were underexpressed based on the reversed-phase chromatogram alone. Studies with standard proteins established the limits of detection to be in the 2.5-5-fmol range. Studies on serum samples showed differences in glycosylation patterns, particularly with respect to sialylation, mannosylation, and fucosylation, in normal, pancreatitis, and cancer sera. By coupling glycoprotein enrichment and fractionation with a microarray platform, we have shown that naturally occurring glycoproteins from human serum can be screened and characterized for different glycan structures, thereby allowing one to do comparative studies that monitor individual glycosylation changes within a glycoproteome representing different biological states. This approach may be useful to identify potential biomarkers in cancer.     

Photocatalytic degradation of Congo Red by hydrothermally synthesized nanocrystalline TiO2 and identification of degradation products by LC-MS

Degradation of Congo Red (CR) dye in aqueous solutions was investigated by means of photocatalysis of TiO2 which was hydrothermally synthesized at 200 degrees C in 2 h, in anatase phase with 8 nm crystallite size. Efficiency of TiO2 in photocatalytic degradation under visible irradiation was studied by investigating the effects of amount of TiO2, irradiation time, initial CR concentration and pH. It was found that complete decolorization is achieved within 30 min of irradiation. Effects of nitrate and sulphate ions and humic acid on the degradation were also tested. The results were compared with Degussa P-25 TiO2 at the same degradation conditions. Degradation products were detected using LC-MS technique. The probable pathways for the formation of degradation products were proposed.     

TiO2改性静电纺复合超滤膜抗污染性能的研究

膜污染是制约超滤膜广泛使用的最重要因素之一,膜污染直接影响到膜的使用寿命及膜的分离性能.本文采用静电纺丝技术制备了PET/PVA纳米纤维复合超滤膜,通过溶剂浸泡处理复合膜,将PVA纳米纤维层溶胀并交联,形成具有抗污染性能的PVA表面致密层结构,所制备的复合纳米纤维超滤膜具有水通量损失率小、通量恢复率高的优点.通过在PVA中添加不同质量分数的TiO2进一步改善膜的亲水性和抗污染性能.使用死端过滤系统过滤10 mg/L腐殖酸溶液,测试结果表明:复合膜的分离性能和抗污染性能在一定范围内随着TiO2的增加而增大.亲水性TiO2的添加能够进一步增强PVA的亲水性,对复合膜抗污染性能的提高有重要作用.但是,TiO2的添加也会增大膜表面的粗糙度,不利于膜抗污染性能的提高,因此,TiO2有一个合宜的添加限度.     

负载型TiO_2超滤膜的制备

以钛酸四异丙酯为原料,采用溶胶-凝胶法制备Ti O2粒子溶胶,以孔径100 nm的管式Al2O3微滤膜为支撑体制备超滤膜,研究了涂膜溶胶中添加剂比例、涂膜次数和涂膜时间对超滤膜完整性的影响。结果表明,调整添加剂比例、涂膜次数和涂膜时间可以消除超滤膜的缺陷;添加剂比例对Ti O2超滤膜的物相没有影响。溶胶中添加剂比例m(Ti O2)∶m(PVA)∶m(HPC)=8∶2∶2,涂膜两次,每次涂膜时间为10~15s时,可以获得无裂纹、无针孔的超滤膜,Ti O2超滤层厚度约1μm,孔径分布于3~8 nm之间,孔隙率42%。     

Screening of nerve agent degradation products by MALDI-TOFMS

A novel method for the rapid screening of degradation products derived from nerve agents by matrix-assisted laser desorption ionization time-of-flight mass spectrometry is described. Five standard products were selected as model compounds, including isopropyl methylphosphonic acid (IMPA), pinacolyl methylphosphonic acid (PMPA), ethyl methylphosphonic acid (EMPA), isobutyl methylphosphonic acid (i-BuMPA), and cyclohexyl methylphosphonic acid (CHMPA), which are degradation products of Sarin (GB), Soman (GD), VX, Russian VX (RVX), and GF, respectively. For comparison, CHCA (alpha-cyano-4-hydroxycinnamic acid) and DCCA (7-(diethylamino)coumarin-3-carboxylic acid) were used as the MALDI-matrix when the third harmonic generation (355 nm) of a Nd:YAG laser and a hydrogen Raman laser (multifrequency laser) were used, respectively. The method permitted the five nerve agent degradation products to be screened rapidly and successfully, suggesting that it has the potential for use as a routine monitoring tool.     

Screening for enzyme inhibitors by surface plasmon resonance combined with mass spectrometry

We have developed a novel strategy to identify enzyme inhibitors that interact directly with their enzyme targets. In the approach, an enzyme is immobilized on a sensor chip, and it is determined whether the immobilized enzyme is still active by incubation with model substrates and mass spectrometric analysis of the products. Putative inhibitors or mixtures containing putative inhibitors are then injected over the sensor chip for binding analysis with surface plasmon resonance. It is then tested whether the bound compounds inhibit the enzymatic activity by subsequent incubation with the model substrate and mass spectrometric analysis. If the bound compound inhibits the enzyme, the inhibitor is eluted from the enzyme and characterized by mass spectrometry. To test the strategy, it has been applied to the well-characterized interaction between trypsin and pure bovine pancreas trypsin inhibitor. Furthermore, fractions of plant extracts were screened for binding to and inhibition of carboxypeptidase B.     

TiO_2/酚酞聚醚砜复合超滤膜的研究

采用浸没沉淀相转化法制备了TiO2/酚酞聚醚砜复合膜,系统地研究了溶剂种类、TiO2添加量等条件对TiO2/PES-C膜结构与性能的影响.结果表明:以DMAc为溶剂时液-液分层速度较慢,得到的超滤膜在添加TiO2前后透过性能均较好,截留率高.与不含纳米TiO2的PES-C膜相比,含TiO2的TiO2/PES-C复合膜的超滤性能、亲水性和抗蛋白污染性都有了显著的改善.当TiO2添加量小于3%时,膜表面Ti元素的含量随着TiO2添加量的增加而增加,其透过性能和抗污染性能也在提高,纳米TiO2/PES-C复合膜具有不对称的断面结构和致密的皮层,亚孔层具有更好的贯通性;当TiO2添加量超过5%时,表面明显分布有大量小孔,其断面结构则表现为皮层变厚,亚孔层消失,只存在指状大孔结构,同时膜的透过性能有所降低.在膜的抗污染试验中,通过牛血清白蛋白溶液连续运行和接触角测定实验表明,TiO2的加入有助于减缓膜在运行过程中通量的衰减,增强了膜的抗蛋白污染性和亲水性.当TiO2水溶液添加量为3%时,TiO2/PES-C复合膜的通量达到最大,亲水性最好,且具有较好的抗蛋白污染性.     

Fuzzy modeling and simulation for lead removal using micellar-enhanced ultrafiltration (MEUF)

In the present paper, a three factor, three-level response surface design based on Box-Behnken design (BBD) was developed for maximizing lead removal from aqueous solution using micellar-enhanced ultrafiltration (MEUF). Due to extremely complexity and nonlinearity of membrane separation processes, fuzzy logic (FL) models have been driven to simulate MEUF process under a wide range of initial and hydrodynamic conditions. Instead of using mathematical model, fuzzy logic approach provides a simpler and easier approach to describe the relationships between the processing variables and the metal rejection and permeation flux. Statistical values, which quantify the degree of agreement between experimental observations and numerically calculated values, were found greater than 91% for all cases. The results show that predicted values obtained from the fuzzy model were in very good agreement with the reported experimental data.     

LC-MS/MS approach for quantification of therapeutic proteins in plasma using a protein internal standard and 2D-solid-phase extraction cleanup

The bioanalysis of plasma samples generated from in vivo studies of therapeutic proteins is of increasing interesting in the biopharmaceutical industry. The conventional ELISA approach has a long assay development time which can limit use in the early discovery and development of protein-based drugs. In this study, an LC-MS/MS bioassay was developed for the quantification of somatropin and a therapeutic human monoclonal antibody. The assay used bovine fetuin as an internal standard and a two-dimensional solid-phase extraction for the cleanup of the plasma digest. Sample extracts were resolved on an analytical size column using a 6 min LC gradient and analyzed using a triple-quadruple mass spectrometer. The linearity of the assay for somatropin was established from 1 to 1000 microg/mL with accuracy and precision within 15%. This LC-MS approach was also applied to a rat pharmacokinetic study of the therapeutic monoclonal antibody with a lower quantitation limit of 0.5 microg/mL. The LC-MS assay had improved accuracy and precision, and the results from analysis of in vivo study samples showed good agreement with the data obtained with an ELISA. The results from this study indicate that the LC-MS bioassay is a simple and feasible approach for the bioanalysis of therapeutic proteins to support in vivo studies during early drug discovery and development.     

Micellar enhanced ultrafiltration of eosin dye using hexadecyl pyridinium chloride

Surfactant-based separation of toxic eosin dye is studied to estimate the potential of micellar enhanced ultrafiltration (MEUF) using cetyl(hexadecyl) pyridinium chloride (CPC) as the cationic surfactant. The optimum feed CPC concentration is found from the experimental results of pure CPC solution in a batch cell. It is in the range of 10-20 kg/m(3) to have a reasonable permeate flux and lower surfactant concentration in the permeate. Selecting 10 kg/m(3) as the feed surfactant concentration, MEUF experiments are conducted to study the retention characteristics of eosin dye in the continuous cross flow system. The effects of operating conditions, i.e., feed dye concentration, operating pressure and cross flow rate on the permeate flux and observed retention of dye are investigated.     

Method for internal standard introduction for quantitative analysis using on-line solid-phase extraction LC-MS/MS

A novel approach for on-line introduction of internal standard (IS) for quantitative analysis using LC-MS/MS has been developed. In this approach, analyte and IS are introduced into the sample injection loop in different steps. Analyte is introduced into the injection loop using a conventional autosampler (injector) needle pickup from a sample vial. IS is introduced into the sample injection loop on-line from a microreservoir containing the IS solution using the autosampler. As a result, both analyte and IS are contained in the sample loop prior to the injection into the column. Methodology allowed to reliably introduce IS and demonstrated injection accuracy and precision comparable to those obtained using off-line IS introduction (i.e., IS and analyte are premixed before injection) while maintaining chromatographic parameters (i.e., analyte and IS elution time and peak width). This new technique was applied for direct analysis of model compounds in rat plasma using on-line solid-phase extraction (SPE) LC-MS/MS quantification. In combination with on-line SPE, IS serves as a surrogate IS and compensates for signal variations attributed to sample preparation and instrumentation factors including signal suppression. The assays yielded accuracy (85-119%), precision (2-16%), and analyte recovery comparable to those obtained using off-line IS introduction. Furthermore, on-line IS introduction allows for nonvolumetric sample (plasma) collection and direct analysis without the need of measuring and aliquoting a fixed sample volume prior to the on-line SPE LC-MS/MS analysis. Therefore, this methodology enables direct sample (plasma) analysis without any sample manipulation and preparation.