High throughput screening of gene functions in mammalian cells using reversely transfected cell arrays: review and protocol

Reversely transfected cell microarrays (RTCM) have been introduced as a method for parallel high throughput analysis of gene functions in mammalian cells. Hundreds to thousands of different recombinant DNA or RNA molecules can be transfected into different cell clusters at the same time on a single glass slide with this method. This allows either the simultaneous overexpression or--by using the recently developed RNA interference (RNAi) techniques--knockdown of a huge number of target genes. A growing number of sophisticated detection systems have been established to determine quantitatively the effects of the transfected molecules on the cell phenotype. Several different cell types have been successfully used for this procedure. This review summarizes the presently available knowledge on this technique and provides a laboratory protocol.     

High throughput screening arrays of rhodium and iridium complexes as catalysts for intramolecular hydroamination using parallel factor analysis

Parallel factor analysis (PARAFAC) was used to analyze data from the high throughput screening of an array of organometallic rhodium and iridium complexes as catalysts for the intramolecular hydroamination of 2-(2-phenylethynyl)aniline to give 2-phenylindole. The progress of the hydroamination reactions was monitored using UV-visible spectroscopy. The overlapped UV-visible spectra of the mixture of starting material, product and solvent in the samples taken at different times were deconvoluted using PARAFAC. Unique PARAFAC models led to close approximations of the actual UV-visible spectra of the compounds in the mixture. The performance of the catalysts was then compared by estimating the final concentration of the starting material and product using PARAFAC loadings. A library of 63 complexes generated in situ was examined in a single experiment using this methodology. The complexes were generated from combinations of seven ligands (bis(N-methyl2-imidazolyl)methane, bis(1-pyrazolyl)methane, 1,10-phenanthroline, N,N'-bis(p-tolyl)diazabutadiene, N,N'-bis(p-tolyl)1,2-dimethyldiazabutadiene, N,N'-bis(mesityl)1,2-dimethyldiazabutadiene and bis(2,4,6-trimethylphenylimino)acenapthene) and nine metal precursors ([Ir(COD)Cl](2) (COD = 1,5-cyclooctadiene), [Ir(CO)(2)Cl](n), [Ir(COE)(2)Cl](2), [IrCp*Cl(2)](2) (Cp* = 1,2,3,4,5-pentamethylcyclopentadiene), [Rh(COD)Cl](2), [Rh(CO)(2)Cl](2), [Rh(COE)(2)Cl](2), [RhCp*Cl(2)](2) and [RhCpCl(2)](2)) (Cp = cyclopentadiene)). The proposed method can be used for the fast screening of arrays of metal complexes for identifying effective catalysts, providing information that can augment traditional methods used for the analysis of catalyzed reactions.     

Evaluation of a method for high throughput solubility determination using a multi-wavelength UV plate reader

Aqueous solubility is a critical physicochemical property and must be addressed early during drug discovery research. Due to the difficulty in accurately predicting aqueous solubility in silico, high throughput experimental determination of aqueous solubility is in great demand. This study evaluates a method using a multi-wavelength UV plate reader and disposable 96-well UV plates for fast solubility determination. It was demonstrated that this method has the sensitivity and reproducibility to effectively determine solubility as low as 1 micro M. Excellent correlation (R>0.97) was observed between the solubility determined using the UV reader method and the HPLC method over the range of 1-1000 micro M for a diverse set of pharmaceutical compounds. In addition to excellent sensitivity and reproducibility, the UV plate reader method also offers the flexibility of being able to determine thermodynamic solubility in the presence or absence of dimethyl sulfoxide, which is a solvent widely used for combinatorial compounds during high throughput screening.     

High throughput genetic screening for the detection of hereditary non-polyposis colon cancer (HNPCC) using capillary electrophoresis

Approximately 5-10% of all colorectal carcinomas arise from cancer predisposition syndromes caused by heterozygote germline mutations in post-replicative DNA mismatch repair (MMR) genes. In contrast to gastrointestinal polyposis syndromes, carcinomas in these patients do not occur on the background of increased numbers of polyps and hence are refered to as hereditary non-polyposis colorectal cancers (HNPCC). Six different MMR genes, MSH2, MSH3, MSH6, MLH1, MLH3 and PMS2, have been identified in the human genome. In the majority of HNPCC patients, heterozygote germline mutations are present in the MSH2 or MLH1 gene. Detection of mutations by conventional sequencing technology is expensive and labor intensive due to the complex intron and/or exon structures. In this study, we therefore have explored whether capillary electrophoresis-based single strand conformation polymorphism (SSCP-CE) provides a reliable means for mutation screening. We have tested different MLH1 mutations in exons 9 and 16 and find that SSCP-CE produces reliable electrophoretic patterns that allow recognition of wild-type alleles, microdeletions and point mutations. In summary, SSCP-CE provides a rapid, automated, and cost-effective technology for MSH2 and MLH1 mutation screening and will facilitate genetic diagnostics for HNPCC patients.     

Micro-well arrays for 3D shape control and high resolution analysis of single cells

In addition to rigidity, matrix composition, and cell shape, dimensionality is now considered an important property of the cell microenvironment which directs cell behavior. However, available tools for cell culture in two-dimensional (2D) versus three-dimensional (3D) environments are difficult to compare, and no tools exist which provide 3D shape control of single cells. We developed polydimethylsiloxane (PDMS) substrates for the culture of single cells in 3D arrays which are compatible with high-resolution microscopy. Cell adhesion was limited to within microwells by passivation of the flat upper surface through 'wet-printing' of a non-fouling polymer and backfilling of the wells with specific adhesive proteins or lipid bilayers. Endothelial cells constrained within microwells were viable, and intracellular features could be imaged with high resolution objectives. Finally, phalloidin staining of actin stress fibers showed that the cytoskeleton of cells in microwells was 3D and not limited to the cell-substrate interface. Thus, microwells can be used to produce microenvironments for large numbers of single cells with 3D shape control and can be added to a repertoire of tools which are ever more sought after for both fundamental biological studies as well as high throughput cell screening assays.     

Integrity profiling of high throughput screening hits using LC-MS and related techniques

Integrity profiling of HTS hits is valuable for verification of the hit identity and purity. This provides early discovery researchers with more confident SAR theories. Methodology for integrity profiling of HTS hits must be high throughput, consume little material, and selectively provide structure-based data. Analytical techniques that can be utilized for integrity profiling methods are reviewed for their appropriateness in sample preparation, component separation, detection, purity quantitation, identity confirmation, and follow-up.     

Electronic properties of Ag nanoparticle arrays. A Kelvin probe and high resolution XPS study

The electronic properties of citrate stabilised Ag nanoparticles with sizes ranging from 4 to 35 nm were investigated by the Kelvin probe method and high resolution XPS. Two and three dimensional assemblies of the particles were prepared by electrostatic adsorption from aqueous solution onto poly-l-lysine modified surfaces. The work function of the Ag particles increased from 5.29 +/- 0.05 to 5.53 +/- 0.05 eV as the particle size decreased. These values are approximately 0.8 eV higher than for clean polycrystalline Ag surfaces. The origin of these remarkable high work functions cannot be explained in terms of either citrate induced changes in the surface dipole or image forces in the confined metallic domains. High resolution XPS spectra of the Ag 3d(5/2) core level were characterised by broad bands and a 0.4 eV shift towards lower binding energies for the smallest particles. Comparisons with reported studies on extended Ag surfaces indicate that as-grown particles exhibit partially oxidised surfaces. The behaviour of the work function further suggests that the strength of the Ag-O bonding increases with decreasing particle sizes. These findings are highly relevant to the interpretation of the catalytic properties of Ag nanoparticles.     

The determination of arsenic and selenium in standard reference materials using sector field ICP-MS in high resolution mode

Sector field ICP-MS was used to analyse As and Se in a range of standard reference materials (NIST 1643d Water, NIST 1573a Tomato Leaves, NIST 1566a Oyster Tissue, NIST 2704 Buffalo River Sediment and Bio-Rad Reference Urine Level 2). A spectral resolution of m/Δm = 7500 enabled ⁷⁵As and ⁷⁷Se to be separated from problematic ArCl interferences. Following microwave acid digestion, solid samples were typically diluted 1 + 99 prior to analysis, while the urine sample was diluted 1 + 9. The water sample was analysed undiluted and diluted 1 + 9. Despite near baseline spectral separation, ⁷⁵As and ⁷⁷Se were still found to be influenced by ArCl at high Cl concentrations, the effect being most pronounced for ⁷⁷Se. When necessary ⁸²Se was also monitored to determine the accuracy of the ⁷⁷Se results. Detection limits (LOD, based on 3σ of 10 replicates) for ⁷⁵As, ⁷⁷Se and ⁸²Se in ultra-pure water, 1% (w/w) HNO₃ and 1% (w/w) HCl were ～?0.1, ～?0.2 and ～?0.5 ng g^–1, respectively. Although signal intensities when using high resolution were ～?1% of that found when using low resolution mode (m/Δm = 300), measured As concentrations and certified values were found to agree to within ± 11% for all samples analysed. The concentration of Se in NIST 1566a Oyster Tissue, NIST 2704 Buffalo River Sediment and Bio-Rad Reference Urine were found to be in agreement with certified values to within ± 15– 20%, as measured by ⁷⁷Se. However, closer agreement (± 5%) was found when these samples were analysed using ⁸²Se. The Se concentration in NIST 1643d Water was found to agree to within ± 5% of the certified value (depending on dilution factor). Due to the low concentration of Se in NIST 1573a Tomato Leaves, quantitation was not possible (below LOQ, 10σ). As a consequence of the lower ion transmission when using resolution 7500, analytical precisions were found to be elevated over that normally observed using low resolution mode, typically ± 5–20% (depending on analyte concentration and isotopic abundance).     

The determination of arsenic and selenium in standard reference materials using sector field ICP-MS in high resolution mode

Sector field ICP-MS was used to analyse As and Se in a range of standard reference materials (NIST 1643d Water, NIST 1573a Tomato Leaves, NIST 1566a Oyster Tissue, NIST 2704 Buffalo River Sediment and Bio-Rad Reference Urine Level 2). A spectral resolution of m/Δm = 7500 enabled ⁷⁵As and ⁷⁷Se to be separated from problematic ArCl interferences. Following microwave acid digestion, solid samples were typically diluted 1 + 99 prior to analysis, while the urine sample was diluted 1 + 9. The water sample was analysed undiluted and diluted 1 + 9. Despite near baseline spectral separation, ⁷⁵As and ⁷⁷Se were still found to be influenced by ArCl at high Cl concentrations, the effect being most pronounced for ⁷⁷Se. When necessary ⁸²Se was also monitored to determine the accuracy of the ⁷⁷Se results. Detection limits (LOD, based on 3σ of 10 replicates) for ⁷⁵As, ⁷⁷Se and ⁸²Se in ultra-pure water, 1% (w/w) HNO₃ and 1% (w/w) HCl were ∼ 0.1, ∼ 0.2 and ∼ 0.5 ng g^–1, respectively. Although signal intensities when using high resolution were ∼ 1% of that found when using low resolution mode (m/Δm = 300), measured As concentrations and certified values were found to agree to within ± 11% for all samples analysed. The concentration of Se in NIST 1566a Oyster Tissue, NIST 2704 Buffalo River Sediment and Bio-Rad Reference Urine were found to be in agreement with certified values to within ± 15– 20%, as measured by ⁷⁷Se. However, closer agreement (± 5%) was found when these samples were analysed using ⁸²Se. The Se concentration in NIST 1643d Water was found to agree to within ± 5% of the certified value (depending on dilution factor). Due to the low concentration of Se in NIST 1573a Tomato Leaves, quantitation was not possible (below LOQ, 10σ). As a consequence of the lower ion transmission when using resolution 7500, analytical precisions were found to be elevated over that normally observed using low resolution mode, typically ± 5–20% (depending on analyte concentration and isotopic abundance). Received: 28 December 1998 / Accepted: 9 February 1999     

The determination of six taste and odour compounds in water using Ambersorb 572 and high resolution mass spectrometry.

A method for the analysis of six taste and odour causing compounds in aqueous samples using the granular adsorbent, Ambersorb 572, and gas chromatography-high resolution mass spectrometry (GC-HRMS) has been developed. This method for the determination of geosmin, 2-methylisoborneol (2-MIB), 2-isopropyl-3-methoxypyrazine (IPMP), 2-isobutyl-3-methoxypyrazine (IBMP), 2,3,6-trichloroanisole (236-TCA) and 2,4,6-trichloroanisole (246-TCA) is highly productive [up to 40 samples per day + 23 quality control (QC) samples] and provides rapid (24-48 h) turnaround times. The analytes are extracted from water by the addition of Ambersorb 572 to the sample bottle and rolling for 1 h. The adsorbent is isolated by filtration and allowed to air dry for 1 h. The Ambersorb 572 is transferred to an autosampler vial and the analytes are desorbed into dichloromethane. The extract is analysed by GC-HRMS at 7000 resolving power (RP). Quantification is performed by isotope dilution and internal standard techniques utilizing d3-geosmin, d3-2-MIB, d5-246-TCA and 2-sec-butyl-3-methoxypyrazine (s-BMP). Method precisions of 3.5-5.8% and accuracies of +/- 5.7-8.9% were obtained. Reporting detection limits (RDLs) of 1.0 ng L-1 were obtained for 2-MIB, geosmin, IPMP and IBMP, while RDLs of 2.0 ng L-1 were obtained for 236-TCA and 246-TCA.     

Rapid and high throughput separation technologies--steady state recycling and supercritical fluid chromatography for chiral resolution of pharmaceutical intermediates

The SSR and SFC techniques were used for the enantiomeric resolution of three pharmaceutical intermediates at various sample scales. The separation conditions, the sample purities and yields, the productivities and the solvent consumptions were discussed in three case studies in this paper. In case (I), the SSR process was used for a low selectivity resolution of 2.0 kg of pharmaceutical intermediate. By using this separation process, a productivity of 750 g racemate/kg stationary phase/day was achieved, while solvent usage was minimized ( approximately 200 l/kg racemate). Case (II) pertained to the effectiveness of the SSR process. Productivity using SSR techniques increased by a factor of 4.5, while solvent usage decreased by a factor of 4.1 when compared to the productivity and solvent usage of batch HPLC. Case (III) compared SFC purification to HPLC purification. The SFC process was more effective in terms of an increase in productivity and a reduction in solvent usage. Based on these results, it appears that SSR and SFC are very useful choices at the early stage of the drug development for a high throughput and a rapid turn around of samples.     

High throughput online sequential extraction of natural rare earth elements and determination by mass spectrometry

At the beginning of rare earth industry,several days are normally required for characterization of rare earth elements(REEs)fractionation in ore samples.Herein,rapid fractionation analysis of 15 REEs and accompanied metal(Fe,Mn,etc.)in ore samples has been achieved within 1 h using ICP-MS with a homemade device for online sequential solvent extraction.As a result,five fractionations for REEs occurrences,i.e.,water soluble,exchangeable,reducible,oxidizable and crystalline,have been identified,offering chemical insights which not only reveal the formation mechanism of REEs ores but also show great implications for guiding the exploitation and separation of REEs.In comparison with conventional methods,the present approach significantly shortened the analysis time(1 h vs.~80 h)and reduced the sample consumption(1.0 mg vs.5.0 g)with high recovery(>95%),providing a useful platform for the rapid quantitative fractionation analysis of REEs in complexed samples such as ore and fossils.     

High resolution gas chromatographic determination of diterpenic alcohols and sterols in coffee lipids

Summary The composition of the unsaponifiable fraction of coffee lipids extracted before and after roasting was determined in two coffee types (Arabica and Robusta) of different geographic origin. Component identification was performed by gas chromatography-mass spectrometry. Large amounts of diterpene mono- and di-alcohols were found in both varieties; cafestol, kahweol and 16-O-methylcafestol were identified. Other components that were partly generated during roasting were also identified; these compounds seem to arise from the dehydration of cafestol and dehydrocafestol.     

High resolution gas chromatographic determination of diterpenic alcohols and sterols in coffee lipids

Summary The composition of the unsaponifiable fraction of coffee lipids extracted before and after roasting was determined in two coffee types (Arabica and Robusta) of different geographic origin. Component identification was performed by gas chromatography-mass spectrometry. Large amounts of diterpene mono-and di-alcohols were found in both varieties; cafestol, kahweol and 16-O-methylcafestol were identified. Other components that were partly generated during roasting were also identified; these compounds seem to arise from the dehydration of cafestol and dehydrocafestol.     

A high throughput approach for determination of dermorphin in human urine using liquid chromatography–mass spectrometry for doping control purposes

Dermorphin is a peptide with analgesic actions similar to morphine, but with greater effect and less potential to cause tolerance. The use of dermorphin has been documented in race horses, and its use in humans has already been reported. Considering the potential advantages from the use of dermorphin over morphine, a method to monitor it, and its main metabolite dermorphin (1‐4) in humans becomes necessary for doping control. Here, we present two orthogonal methods for this purpose: a high‐throughput liquid chromatography coupled to high‐resolution mass spectrometry (HRMS) as an initial testing procedure and liquid chromatography–tandem mass spectrometry (MS/MS) in the selected reaction monitoring (SRM) acquisition mode for a confirmation procedure. For urine samples, pretreatment through a mixed‐mode weak cation‐exchange solid‐phase extraction emerged as an effective approach to extract peptides from the biological sample. For the HRMS analysis, a full‐MS scan acquisition mode was selected to detect the exact masses of dermorphin and dermorphin (1‐4) at m/z 803.37226 and 457.20816, respectively. The SRM method used in the MS/MS confirmation protocol presented high specificity and sensitivity. The selected product ions for dermorphin were 602.2, 202.1 and 574.3 and for dermorphin (1‐4) were 207.1, 223.1, and 235.1. Both methods were evaluated for specificity, repeatability, carryover, matrix effects, and recovery. No carryover and matrix effects were detected. The limit of detection for initial testing procedure and the limit of identification for confirmation procedure was 2.5 ng/ml. Also, specificity and robustness were acceptable for the application. Together, the developed methods proved to be efficient for the analysis of dermorphin and metabolite for human doping control purpose.     

大流量采样高分辨气相色谱/高分辨质谱法测定大气中的多氯联苯和多溴联苯醚

建立了大流量空气采样高分辨气相色谱/高分辨质谱(HRGC/HRMS)同时分析测定大气样品中多氯联苯(PCBs)和多溴联苯醚(PBDEs)的分析方法。结果表明在采样过程中污染物没有发生穿透。通过添加 13C同位素标准物质进行评价,PCBs和PBDEs的加标回收率分别为60.7%～121.4%和69.9%～140.4%,均符合美国环保署相关方法的要求。PCBs和PBDEs的方法检出限分别低于0.019 pg/m3和0.189 pg/m3;色谱分离效果良好,可以满足大气样品中PCBs和PBDEs的监测需要。     

Trace determination of airborne polyfluorinated iodine alkanes using multisorbent thermal desorption/gas chromatography/high resolution mass spectrometry

A novel gas chromatography/high resolution mass spectrometry method coupled with multisorbent thermal desorption cartridges has been developed for the determination of volatile neutral polyfluorinated iodine alkanes (PFIs) in airborne samples. It allows, for the first time, simultaneous analysis of four mono-iodized perfluorinated alkanes, three diiodized perfluorinated alkanes and four mono-iodized polyfluorinated telomers in ambient air samples. 3.75 L air sample was passed through a sorbent tube packed with 150 mg of Tenax TA and 200 mg of Carbograph 1TD for analyte adsorption. Important factors during the analysis procedures, such as safe sampling volume, air sampling rate, analyte desorption and transfer strategies, were optimized and good thermal desorption efficiencies were obtained. The method detection limit (MDL) concentration ranged from 0.04 pg/L for 1H,1H,2H,2H-perfluorododecyl iodide to 1.2 pg/L for perfluorohexyl iodide, and instrument response of a seven-point calibration was linear in the range of 10–1000 pg. Travel spike recoveries ranged from 83% to 116%. Small variabilities of less than 36% were obtained near the MDLs and the differences between triplicates were even smaller (2.1–7.3%) at 200 pg spiked level. The method was successfully applied to analyze ambient air samples collected near a point source, and five PFIs were identified (10.8–85.0 pg/L), with none of the analytes detectable at the background site.     

Discovery and evaluation of highly active imidotitanium ethylene polymerisation catalysts using high throughput catalyst screening

A family of ca. 50 imidotitanium precatalysts [Ti(NR)(Me(3)[9]aneN(3))Cl(2)](R = alkyl or aryl; Me(3)[9]aneN(3)= 1,4,7-trimethyltriazacyclononane) were prepared in good yields using semi-automated procedures; high-throughput screening techniques identified seven highly active ethylene polymerisation precatalysts with activities in the range ca. 3 400 to 10 000 kg(PE) mol(-1) h(-1) bar(-1).     

Separation of enantiomers of ephedrine by capillary electrophoresis using cyclodextrins as chiral selectors: comparative CE, NMR and high resolution MS studies

The enantiomer migration order (EMO) of ephedrine was investigated in the presence of various CDs in CE. The molecular mechanisms of chiral recognition were followed for the ephedrine complexes with native α- and β-CD and heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD (HDAS-β-CD) by CE, NMR spectroscopy and high-resolution MS. Minor structural differences were observed between the complexes of ephedrine with α- and β-CD although the migration order of enantiomers was opposite when these two CDs were applied as chiral selectors in CE. The EMO was also opposite between β-CD and HDAS-β-CD. Significant structural differences were observed between ephedrine complexes with the native CDs and HDAS-β-CD. The latter CD was advantageous as chiral CE selector not only due to its opposite electrophoretic mobility compared with that of the cationic chiral analyte, but also primarily due to its enhanced chiral recognition ability towards the enantiomers of ephedrine.     

Multi-elemental analysis of marine sediment reference material MESS-3: one-step microwave digestion and determination by high resolution inductively coupled plasma-mass spectrometry (HR-ICP-MS)

Single-step microwave digestion methods using four different acidic dissolving reagents were investigated with the main objective to obtain a simple, rapid method for multi-elemental analysis of marine sediment samples by high resolution inductively coupled plasma-mass spectrometry (HRICP-MS), especially for environmental studies. Closed-vessels microwave digestion procedures were carried out using the reagents as follows: HNO₃, mixture of HNO₃-HCl (φ _r = 9: 1), aqua regia, and mixture of HNO₃-HCl-HF (φ _r = 8: 1: 1). The elements analyzed were: Al, As, Ba, Be, Bi, Cd, Co, Cr, Cu, Fe, K, Li, Mn, Mo, Na, Ni, Pb, Sb, Sn, Sr, Ti, Tl, V, and Zn. Good recoveries were established for many of the metals analyzed even when only nitric acid was employed. However, for a good recovery of Sb and Tl, aqua regia should be employed.