High-throughput mass spectrometry screening for inhibitors of phosphatidylserine decarboxylase

A high-throughput mass spectrometry assay to measure the catalytic activity of phosphatidylserine decarboxylase (PISD) is described. PISD converts phosphatidylserine to phosphatidylethanolamine during lipid synthesis. Traditional methods of measuring PISD activity are low throughput and unsuitable for the high-throughput screening of large compound libraries. The high-throughput mass spectrometry assay directly measures phosphatidylserine and phosphatidylethanolamine using the RapidFiretrade mark platform at a rate of 1 sample every 7.5 s. The assay is robust, with an average Z' value of 0.79 from a screen of 9920 compounds. Of 60 compounds selected for confirmation, 54 are active in dose-response studies. The application of high-throughput mass spectrometry permitted a high-quality screen to be performed for an otherwise intractable target.     

Improved screening method for beta-blockers in urine using solid-phase extraction and capillary gas chromatography—mass spectrometry

An improved screening method for beta-blockers in urine is proposed, involving enzymatic hydrolysis, solid-phase extraction and capillary gas chromatography—mass spectrometry. Several extraction methods for beta-blockers, such as conventional liquid—liquid and solid-phase extraction procedures, have been evaluated for at least eight beta-blockers. Additionally, the gas chromatographic properties and mass fragmentation of the trimethylsilyl—trifluoroacetyl, trifluoroacetyl and cyclic n-butylboronate derivatives of beta-blockers have been compared and evaluated with respect to their efficiency for screening urine. The resulting screening method proved to be a specific and sensitive procedure, enabling these analytes to be detected and identified up to 48 h after the administration of a dosage, usually encountered in doping cases.     

Determination of 9α,11β-prostaglandin F2 in human urine and plasma by gas chromatography—mass spectrometry

Sensitive and specific assay methods for 9α,11β-prostaglandin F₂ (9α,11β-PGF₂) by gas chromatography—mass spectrometry with electron impact ionization are described. The mass spectrometric assay for 9α,11β-PGF₂ was based on the use of the methyl ester—dimethylisopropylsilyl ether derivative, and pentadeuterated PGF_2α as a convenient internal standard. The calibration graph for 9α,11β-PGF₂ was linear from 5 pg to 100 ng for both the standard and spiked biological samples. The limit of detection was 50 pg/ml for urine and 25 pg/ml for plasma (signal-to-noise RATIO = 2.3). The method was applied to the determination of 9α,11β-PGF₂ in urine and plasma samples from patients with bronchial asthma.     

Quantitative measurement of a new synthetic hetrazepine derivative, BN50730, in human plasma and urine by combined liquid chromatography—negative chemical ionization mass spectrometry using a particle beam interface

A new simple and sensitive assay has been developed for the quantitative measurement of BN50730 at the picomole level in human plasma and urine. The drug and the internal standard (BN50765) were measured by combined liquid chromatography—negative chemical ionization mass spectrometry with methane as the reagent gas. A simple solid—liquid extraction procedure was used to isolate BN50730 from complex biological matrices. Mild operating conditions were required to assay the parent drug with a particle beam interface from Hewlett-Packard. The mass spectrometer was tuned to monitor the intense ion m/z 333, which was generated in the ion source by a dissociative capture process. This assay was performed with 1 ml of plasma or 0.1 ml of urine, and the quantification limit of the method was statistically calculated as 1 ng ml⁻¹. The very low relative standard deviation and mean percentage of error calculated during the different within-day or between-day repeatability assays clearly demonstrate the ruggedness of the technique for the routine determination of BN50730 in the biological fluids. Some preliminary results on the pharmacokinetics of the drug are presented to illustrate the applicability of this new powerful LC—MS method.     

Screening and confirmatory analysis of β-agonists, β-antagonists and their metabolites in horse urine by capillary gas chromatography—mass spectrometry

A method for the screening and confirmatory analysis of β-agonists and -antagonists in equine urine is described. Following initial enzymic hydrolysis, the basic drugs and metabolites are extracted using Clean Screen^® DAU or Bond Elut Certify™ cartridges, and analysed as their trimethylsilyl ether or 2-(dimethyl) silamorpholine derivatives by capillary gas chromatography—mass spectrometry. The method proved to be very sensitive and selective for basic drugs. After administration of therapeutic doses of propranolol, metoprolol, timolol, isoxsuprine and clenbuterol to thoroughbred horses, the parent compound/metabolites could be detected in urine for upto 14–120 h depending on the drug.     

Determination of seven prostanoids in 1 ml of urine by gas chromatography—negative ion chemical ionization triple stage quadrupole mass spectrometry

In an isotope dilution assay, prostaglandin (PG) E₂, 6-keto-PGF_1α, thromboxane (Tx) B₂ and their metabolites PGE-M (11α-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid), 2,3-dinor-6-keto-PGF_1α, 2,3-dinor-TxB₂ and 11-dehydro-TxB₂ were determined in urine by gas chromatography—triple stage quadrupole mass spectrometry (GC—MS—MS). After addition of deuterated internal standards, the prostaglandins were derivatized to their methoximes and extracted with ethyl acetate—hexane. The sample was further derivatized to the pentafluorobenzylesters and purified by thin-layer chromatography (TLC). Three zones were scraped from the TLC plate. The prostanoid derivatives were converted to their trimethylsilyl ethers and the products were quantified by GC—MS—MS. In each run, two or three prostanoids were determined.     

Simultaneous determination of mevalonate and 7α-hydroxycholesterol in human plasma by gas chromatography—mass spectrometry as indices of cholesterol and bile acid biosynthesis

A very sensitive and specific method for the simultaneous determination of mevalonate and 7α-hydroxycholesterol in human plasma is described. The assay is based on isotope dilution mass spectrometry: the extracts from plasma were treated with benzylamine followed by dimethylethylsilylimidazole, then the resulting dimethylethylsilyl ether derivatives of mevalonylbenzylamide and 7α-hydroxycholesterol were determined by gas chromatography—mass spectrometry using high-resolution selected-ion monitoring. Simple regression analysis revealed significant correlations between the plasma level of mevalonate and the hepatic activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34) (r = 0.83, P < 0.01) and between the plasma level of free 7α-hydroxycholesterol and the hepatic activity of cholesterol 7α-hydroxylase (EC 1.14.13.7) (r = 0.76, P < 0.05).     

Screening for lysine-specific demethylase-1 inhibitors using a label-free high-throughput mass spectrometry assay

Posttranslational modifications on the N terminus of histone H3 act in a combinatorial fashion to control epigenetic responses to extracellular stimuli. Lysine-specific demethylase-1 (LSD1) represents an emerging epigenetic target class for the discovery of novel antitumor therapies. In this study, a high-throughput mass spectrometry (HTMS) assay was developed to measure LSD1-catalyzed demethylation of lysine-4 on several H3 substrates. The assay leverages RapidFire chromatography in line with a triple stage quadrupole detection method to measure multiple LSD1 substrate and product reactions from an assay well. This approach minimizes artifacts from fluorescence interference and eliminates the need for antibody specificity to methylated lysines. The assay was robust in a high-throughput screen of a focused library consisting of more than 56,000 unique chemical scaffolds with a median Z′ of 0.76. Validated hits from the primary screen were followed up by successive rounds of virtual and HTMS screening to mine for related structures in a parent library consisting of millions of compounds. The screen resulted in the rapid discovery of multiple chemical classes amenable to medicinal chemistry optimization. This assay was further developed into a generic platform capable of rapidly screening epigenetic targets that use the N-terminal tail of histone H3 as a substrate.     

Modeling the performance of immobilized α-chymotrypsin catalyzed peptide synthesis in acetonitrile medium

A model was developed which describes simultaneous reaction and internal diffusion for kinetically controlled, immobilized α-chymotrypsin-catalyzed, oligopeptide synthesis in acetonitrile medium. The model combines the equations that describe the intrinsic kinetics of four different reactions and the physical characteristics of three different support materials, as determined experimentally, to predict the apparent initial activity and nucleophile selectivity of the immobilized biocatalyst. The model is able to predict reasonably well the experimentally observed initial rate and nucleophile selectivity vs. enzyme loading profiles. The reduction in observed initial rate with enzyme loading when fast reactions are carried out with α-chymotrypsin immobilized on celite, and the larger influence of mass transfer limitations on the initial reaction rates than on nucleophile selectivities are correctly predicted by the numerical calculations. The model is general in terms of its application to other systems — enzymes, reactions, support materials and/or kinetic schemes — as long as the intrinsic kinetics and the characteristics of the enzyme and support material are known.     

Blood group glycosphingolipid expression in kidney of an individual with the rare blood group A1 Le(a−b+) p phenotype: absence of blood group structures based on the globoseries

Total neutral glycolipid fractions were isolated from kidney and ureter tissue obtained at autopsy of an individual of the rare blood group A₁ Le(a–b+) p. The amount of glycolipids isolated were 3.7 and 2.5 mg g^–1 dry tissue weight for the kidney and ureter tissue, which is in the range of reference blood group P kidneys. Part of the kidney glycolipid fraction was subfractionated by HPLC. Glycolipid compounds were structurally characterized by thin-layer chromatography (chemical detection and immunostaining with monoclonal antibodies), proton NMR spectroscopy and mass spectrometry. Globotriaosyl- and globotetraosyl-ceramides, which are the major compounds in kidneys of P individuals, were absent in the p kidney, and a comparatively increased amount of monoglycosyland lactosylceramides was found. A shift to longer fatty acyl chains in the ceramide part of lactosylceramides was noted. Elongated globoseries compounds with five to seven sugar residues, including the blood group A type 4 chain structure, were lacking. A slight increase in neolactotetraosyl- and blood group X pentaglycosyl-ceramides was noticed. The study confirms an enzymatic block in the conversion of lactosylceramide to elongated globoseries compounds in the kidney tissue similar to that of erythrocytes of p individuals.Abbreviations: for blood group glycolipid antigens the short hand designation stands for: blood group — number of sugar residues — type of carbohydrate chain. Thus A-7-4 means a blood group A heptaglycoconjugate on a type 4 chain. The sugar types are abbreviated for mass spectrometry to Hex for hexose, HexNAc forN-acetylhexosamine and dHex for deoxyhexose. HPLC, high-performance liquid chromatography; HPTLC, high performance thin layer chromatography; EI, electron impact ionisation; LSI, liquid secondary ion; MS, mass spectrometry; NMR, nuclear magnetic resonance.     

Determination of very-long-chain fatty acids in plasma by a simplified gas chromatographic—mass spectrometric procedure

The concentration of very-long-chain fatty acids (VLCFA) (straight chain, more than 22 carbon atoms) in plasma or in cultured fibroblasts is one of the most important diagnostic criteria for the diagnosis of the peroxisomal disorders. A sensitive method for VLCFA assay in plasma, using small sample volume and a simplified procedure, is described. After adequate extraction and derivatization, methyl esters of VLCFA are separated, identificated and quantified by gas chromatography—mass spectrometry (GC—MS). The method is sensitive, reproducible, accurate and relatively simple. GC—MS equipment used for routine organic acid analysis can be used.     

Improved assay for the quantification of 11-dehydrothromboxane B2 by gas chromatography—mass spectrometry

Endogenous thromboxane production is best assessed by the measurement of its excreted metabolites, of which 11-dehydrothromboxane B₂ (11-dehydro-TxB₂) is most abundant. Gas chromatographic—mass spectrometric assays have been developed for this compound but suffer from the presence of co-eluting impurities which make the measurement of 11-dehydro-TxB₂ difficult. Furthermore, these assays are often time-consuming. We now report a modified assay for the measurement of this compound employing gas chromatography—mass spectrometry which alleviates the problem of co-eluting impurities primarily through modification of extraction and chromatographic methods. Furthermore, the time to complete the assay is significantly shortened. It is adaptable to both urine and plasma. Precision of the assay is ± 7% and accuracy is 90%. The lower limit of sensitivity in urine is approximately 20 pg/mg creatinine. Normal levels of urinary excretion of this metabolite were found to be 370 ± 137 pg/mg creatinine (mean ± 1 S.D.) and normal plasma levels were found to be 1.5 ± 0.4 pg/ml (mean ± 1 S.D.). Urinary excretion of 11-dehydro-TxB₂ is markedly altered in situations associated with abnormalities in thromboxane generation when quantified using this assay. Thus, this assay provides a sensitive and accurate method to assess endogenous thromboxane production and to further explore the role of this compound in human disease.     

High Resolution Melting Analysis: A Rapid Screening and Typing Tool for Common β-Thalassemia Mutation in Chinese Population

β-thalassemia is a common inherited disorder worldwide including southern China, and at least 45 distinct β-thalassemia mutations have been identified in China. High-resolution melting (HRM) assay was recently introduced as a rapid, inexpensive and effective method for genotyping. However, there was no systemic study on the diagnostic capability of HRM to identify β-thalassemia. Here, we used an improved HRM method to screen and type 12 common β-thalassemia mutations in Chinese, and the rapidity and reliability of this method was investigated. The whole PCR and HRM procedure could be completed in 40 min. The heterozygous mutations and 4 kinds of homozygous mutations could be readily differentiated from the melting curve except c.-78A>G heterozygote and c.-79A>G heterozygote. The diagnostic reliability of this HRM assay was evaluated on 756 pre-typed genomic DNA samples and 50 cases of blood spots on filter paper, which were collected from seven high prevalent provinces in southern China. If c.-78A>G heterozygote and c.-79A>G heterozygote were classified into the same group (c.-78&79 A>G heterozygote), the HRM method was in complete concordance with the reference method (reverse dot blot/DNA-sequencing). In a conclusion, the HRM method appears to be an accurate and sensitive method for the rapid screening and identification of β-thalassemia mutations. In the future, we suggest this technology to be used in neonatal blood spot screening program. It could enlarge the coverage of β-thalassemia screening program in China. At the same time, its value should be confirmed in prospectively clinical and epidemiological studies.     

Thermospray and particle beam liquid chromatographic—mass spectrometric analysis of coumarin anticoagulants

Positive ion mass spectra were obtained from several coumarin oral anticoagulants (phenprocoumon, warfarin, acenocoumarol and dicoumarol) and derivatives by liquid chromatography—thermospray mass spectrometry (LC—TSP-MS) and liquid chromatography—electron impact mass spectrometry (LC—EI-MS) to assess the use of LC—MS methods for the determination of these compounds in biological materials. LC—TSP mass spectra showed a single [M + 1]⁺ ion with no fragmentation; LC—EI mass spectra showed fragment ions which were similar in mass and relative intensities to those obtained by conventional EI-MS. These data should serve as a basis for the development of LC—MS methods for the qualitative and quantitative analysis of coumarin anticoagulants in biological samples. LC—TSP-MS was applied to the determination of phenprocoumon in a plasma extract from an anticoagulated patient.     

BeeDoctor,a Versatile MLPA-Based Diagnostic Tool for Screening Bee Viruses

The long-term decline of managed honeybee hives in the world has drawn significant attention to the scientific community and bee-keeping industry. A high pathogen load is believed to play a crucial role in this phenomenon, with the bee viruses being key players. Most of the currently characterized honeybee viruses (around twenty) are positive stranded RNA viruses. Techniques based on RNA signatures are widely used to determine the viral load in honeybee colonies. High throughput screening for viral loads necessitates the development of a multiplex polymerase chain reaction approach in which different viruses can be targeted simultaneously. A new multiparameter assay, called “BeeDoctor”, was developed based on multiplex-ligation probe dependent amplification (MLPA) technology. This assay detects 10 honeybee viruses in one reaction. “BeeDoctor” is also able to screen selectively for either the positive strand of the targeted RNA bee viruses or the negative strand, which is indicative for active viral replication. Due to its sensitivity and specificity, the MLPA assay is a useful tool for rapid diagnosis, pathogen characterization, and epidemiology of viruses in honeybee populations. “BeeDoctor” was used for screening 363 samples from apiaries located throughout Flanders; the northern half of Belgium. Using the “BeeDoctor”, virus infections were detected in almost eighty percent of the colonies, with deformed wing virus by far the most frequently detected virus and multiple virus infections were found in 26 percent of the colonies.     

pH dependence and thermostability of lipases from cultures from the ARS culture collection

Summary Previously we used a simple, sensitive agar plate method to screen lipase activity from 1229 selected cultures including 508 bacteria, 479 yeasts, 230 actinomycetes and 12 fungi covering many genera and species. About 25% of the cultures tested were lipase-positive. These lipase-positive strains were further classified as good, moderate or weak enzyme producers. We have expanded our screening method to focus specifically on the pH dependence and thermostability of these lipase activities. The lipases exhibited various pH sensitivities and were divided into three groups: (i) lipases which are active at pH 5.5 but not at pH 7.5—produced by 36 bacteria, 23 yeasts and four actinomycetes; (ii) lipases which are active at pH 7.5 but not at pH 5.5—produced by 17 bacteria, four yeasts, two actinomycetes and one fungus; and (iii) lipases which are active at both pH 5.5 and pH 7.5—produced by 112 bacteria, 90 yeasts, 15 actinomycetes and five fungi. By screening at 60°C and pH 9.0, we further identified 50 bacteria and 26 yeasts that produce thermostable alkali-tolerant lipases. Product analyses confirmed our screening results. Lipases with specific pH dependency and thermostability have potential to be developed into industrial enzymes.     

Determination of therapeutic and toxic concentrations of doxepin and loxapine using gas—liquid chromatography with a nitrogen-sensitive detector, and gas chromatography—mass spectrometry of loxapine

A gas—liquid chromatographic procedure is presented for the determination of therapeutic and toxic serum levels of doxepin and loxapine, using a nitrogen—phosphorus-sensitive detector. Amitriptyline is used as the internal standard. The method is accurate, sensitive and specific with no derivatization required prior to analysis. An advantage of the procedure is the small serum sample size needed for analysis and the selectivity and sensitivity of the detector, with the limit of detection being 3 and 2 μg/l for doxepin and loxapine, respectively. Nine cases of doxepin and loxapine misuse are presented. Serum doxepin concentrations ranged from 113 to 439 μg/l, with a loxapine concentration of 192 μg/l observed in one patient. The presence of the tricyclics was identified and confirmed by gas chromatography—mass spectrometry and the mass spectrum of loxapine is reported.     

Mass transfer in fermentation

An essential consideration in the design and operation of commercial fermenters is to ensure adequate mass transfer. The complex composition of fermentation liquids makes it difficult to predict accurately the mass transfer characteristics in large vessels. Here various aspects of mass transfer are discussed and their relationships examined. Strategies for predicting the most important type of mass transfer — between gases and liquids — in large scale fermentations are presented.     

Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC₅₀ for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors.