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At least 37 records · Page 2Linked to original sources
PubMed2006

High-throughput and high-resolution flow cytometry in molded microfluidic devices.

We describe the design, fabrication, and operation of two types of flow cytometers based on microfluidic devices made of a single cast of poly(dimethylsiloxane). The stream of particles or cells injected into the devices is hydrodynamically focused in both transverse and lateral directions, has a uniform velocity, and has adjustable diameter and shape. The cytometry system built around the first microfluidic device has fluorescence detection accuracy comparable with that of a commercial flow cytometer and can analyze as many as 17 000 particles/s. This high-throughput microfluidic device could be used in inexpensive stand-alone cytometers or as a part of integrated microanalysis systems. In the second device, a stream of particles is focused to a flow layer of a submicrometer thickness that allows imaging the particles with a high numerical aperture microscope objective. To take long-exposure, low-light fluorescence images of live cells, the device is placed on a moving stage, which accurately balances the translational motion of particles in the flow. The achieved resolution is comparable to that of still micrographs. This high-resolution device could be used for analysis of morphology and fluorescence distribution in cells in continuous flow.

Flow Cytometry↗
PubMed2006

High-throughput and high-resolution two dimensional mapping of pI and m/z using a microchip in a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer.

We have developed a high-throughput, two-dimensional-mapping (isoelectric point [pI], mass-to-charge ratio [m/z]) method by combining a capillary isoelectric focusing chip sealed with removable resin tape and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Sample proteins are separated in a meandering channel on the chip and immediately frozen. The tape is then removed and the proteins are freeze-dried. The freeze-drying maintains the separation state of the proteins and prevents movement of the sample solution, which can reduce pI resolution. A matrix solution is then applied and mass spectrometry is carried out by laser irradiation. The whole process takes less than 70 min, more than 10 times faster than with two-dimensional, polyacrylamide gel electrophoresis.

Isoelectric Focusing↗
PubMed2002

Protein interactions: two methods for assessment of the reliability of high throughput observations.

High throughput methods for detecting protein interactions require assessment of their accuracy. We present two forms of computational assessment. The first method is the expression profile reliability (EPR) index. The EPR index estimates the biologically relevant fraction of protein interactions detected in a high throughput screen. It does so by comparing the RNA expression profiles for the proteins whose interactions are found in the screen with expression profiles for known interacting and non-interacting pairs of proteins. The second form of assessment is the paralogous verification method (PVM). This method judges an interaction likely if the putatively interacting pair has paralogs that also interact. In contrast to the EPR index, which evaluates datasets of interactions, PVM scores individual interactions. On a test set, PVM identifies correctly 40% of true interactions with a false positive rate of approximately 1%. EPR and PVM were applied to the Database of Interacting Proteins (DIP), a large and diverse collection of protein-protein interactions that contains over 8000 Saccharomyces cerevisiae pairwise protein interactions. Using these two methods, we estimate that approximately 50% of them are reliable, and with the aid of PVM we identify confidently 3003 of them. Web servers for both the PVM and EPR methods are available on the DIP website (dip.doe-mbi.ucla.edu/Services.cgi).

Algorithms↗
PubMedDate not supplied

Purifying the masses: integrating prepurification quality control, high-throughput LC/MS purification, and compound plating to feed high-throughput screening.

In this paper we report using a parallel, four-channel HPLC/MUX/MS purification system, the Purification Factory, to purify thousands of compounds destined for high-throughput screening in a single month. The maximum sample throughput during this 20-workday month was 704 samples/day. Since this purification throughput exceeded the postpurification sample and data handling capabilities provided by commercial solutions, a custom-integrated solution was designed to address these shortcomings. In this paper we detail the key improvements in automation, solvent handling, and sample handling logistics implemented to sustain a mean throughput of 528 samples/day over a multimonth time period.

Autoanalysis↗
PubMed2024

High-Throughput and High-Sensitivity Biomarker Monitoring in Body Fluid by Fast LC SureQuant IS-Targeted Quantitation.

Targeted proteomics methods have been greatly improved and refined over the last decade and are becoming increasingly the method of choice in protein and peptide quantitative assays. Despite the tremendous progress, targeted proteomics assays still suffer from inadequate sensitivity for lower abundant proteins and throughput, especially in complex biological samples. These attributes are essential for establishing targeted proteomics methods at the forefront of clinical use. Here, we report an assay utilizing the SureQuant internal standard-triggered targeted method on a latest generation mass spectrometer coupled with an EvoSep One liquid chromatography platform, which displays high sensitivity and a high throughput of 100 samples per day. We demonstrate the robustness of this method by quantifying proteins spanning six orders of magnitude in human wound fluid exudates, a biological fluid that exhibits sample complexity and composition similar to plasma. Among the targets quantified were low-abundance proteins such at tumor necrosis factor A and interleukin 1-β, highlighting the value of this method in the quantification of trace amounts of invaluable biomarkers that were until recently hardly accessible by targeted proteomics methods. Taken together, this method extends the toolkit of targeted proteomics assays and will help to drive forward mass spectrometry-based proteomics biomarker quantification.

Humans↗
PubMed1999

Natural Products in High Throughput Screening: Automated High-Quality Sample Preparation.

At present, compound libraries from combinatorial chemistry are the major source for high throughput screening (HTS) programs in drug discovery. On the other hand, nature has been proven to be an outstanding source for new and innovative drugs. Secondary metabolites from plants, animals, and microorganisms show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds, often generated in time-consuming and, for the most part, manual processes. Because quality and quantity of the provided samples play a pivotal role in the success of HTS programs, this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of modified Zymark (Hopkinton, MA) RapidTrace® SPE workstations, we developed an easy-to-handle and effective fractionation method that generates high-quality samples from natural origin, fulfilling the requirements for an integration in high throughput drug discovery programs.

Journal Article↗
PubMed2000

Pattern recognition analysis of endogenous cell metabolites for high throughput mode of action identification: removing the postscreening dilemma associated with whole-organism high throughput screening.

Although whole-organism HTS can give clear indications of in vivo activity, typically few clues are given as to the mechanism of action (MOA), and determining the MOA for large numbers of active compounds can be costly and complex-an alternative approach is required. This report demonstrates that it is possible to conduct relatively high throughput MOA characterization of HTS hits utilizing a single sample preparation and analytical method. By monitoring a wide range of endogenous cellular metabolites via (1)H nuclear magnetic resonance spectroscopy, the MOA of herbicides can be predicted using computational methods to compare the metabolite perturbation patterns. Herbicides that induce a characteristic pattern of metabolic perturbation in maize include inhibitors of acetolactate synthase, acetyl co-enzyme A carboxylase, protoporphyrinogen oxidase, 5-enolpyruvylshikimate-3-phosphate synthase, and phytoene desaturase. In soya, photosystem II inhibitors can also be detected, further demonstrating that this method is not limited to inhibitors of enzymes that directly act upon endogenous metabolites, or a single species. The methods, including data analysis, can be readily automated, enabling relatively high throughput MOA elucidation of whole-organism screen hits. Additionally, for compounds with a novel MOA, this approach may lead to MOA identification faster than traditional methods. It is envisaged that application of these data analysis methods to other data types-for example, transcription (mRNA) or translation (protein) profiles-is likely to permit higher throughput with smaller sample requirements, along with ability to discriminate MOAs that are not adequately discriminated based upon endogenous metabolite profiles.

Automation↗
PubMed2005

Discovery and optimization of new chromium catalysts for ethylene oligomerization and polymerization aided by high-throughput screening.

High throughput screening (HTS) of a 205 member Schiff base salicylaldimine ligand library derived from salicylaldehydes bearing bulky ortho-substituents, i.e., 9-anthracenyl, 1,4,5,8-tetramethylanthracenyl or triptycenyl, reacted in-situ with (p-tolyl)CrCl2(thf)3, identified two new classes of highly active chromium based systems for the oligomerization and polymerization of ethylene, respectively. The polymerization system comprises bidentate ortho-substituted anthracenyl Schiff bases bearing small primary or secondary alkyl imine substituents. The oligomerization catalysts are based upon tridentate ortho-triptycenyl-substituted Schiff bases with pyridylmethyl or quinolyl substituents. Validation tests confirmed polymerization productivities of up to 3000 g x mmol(-1)h(-1)bar(-1) for the polymerization catalyst systems while the oligomerization catalysts gave productivities up to 10 000 g x mmol(-1)h(-1)bar(-1). Key catalyst precursors have been characterized by X-ray crystallography.

Journal Article↗
PubMed2000

Identification of differential gene expression by high throughput analysis.

High throughput analysis of differential gene expression is a powerful tool that can be applied to many areas in molecular cell biology, including differentiation, development, physiology, and pharmacology. In recent years, a variety of techniques have been developed to analyze differential gene expression, including comparative expressed sequence tag sequencing, differential display, representational difference analysis, cDNA or oligonucleotide arrays, and serial analysis of gene expression. This review explains the technologies, their scopes, impact on science, as well as their costs and possible limitations. The application of differential display is presented as a tool to identify genes induced by darkness or yellowing process in rice leaves.

Expressed Sequence Tags↗
PubMed2005

Tackling calibration problems of spectroscopic analysis in high-throughput experimentation.

High-throughput experimentation and screening methods are changing work flows and creating new possibilities in biochemistry, organometallic chemistry, and catalysis. However, many high-throughput systems rely on off-line chromatography methods that shift the bottleneck to the analysis stage. On-line or at-line spectroscopic analysis is an attractive alternative. It is fast, noninvasive, and nondestructive and requires no sample handling. The disadvantage is that spectroscopic calibration is time-consuming and complex. Ideally, the calibration model should give reliable predictions while keeping the number of calibration samples to a minimum. In this paper, we employ the net analyte signal approach to build a calibration model for Fourier transform near-infrared measurements, using a minimum number of calibration samples based on blank samples. This approach fits very well to high-throughput setups. With this approach, we can reduce the number of calibration samples to the number of chemical components in the system. Thus, the question is no longer how many but which type of calibration samples should one include in the model to obtain reliable predictions. Various calibration models are tested using Monte Carlo simulations, and the results are compared with experimental data for palladium-catalyzed Heck cross-coupling.

Journal Article↗
PubMed2005

A test of enhancing model accuracy in high-throughput crystallography.

The high throughput of structure determination pipelines relies on increased automation and, consequently, a reduction of time spent on interactive quality control. In order to meet and exceed current standards in model accuracy, new approaches are needed for the facile identification and correction of model errors during refinement. One such approach is provided by the validation and structure-improvement tools of the MOL: PROBITY: web service. To test their effectiveness in high-throughput mode, a large subset of the crystal structures from the SouthEast Collaboratory for Structural Genomics (SECSG) has used protocols based on the MOL: PROBITY: tools. Comparison of 29 working-set and 19 control-set SECSG structures shows that working-set outlier scores for updated Ramachandran-plot, sidechain rotamer, and all-atom steric criteria have been improved by factors of 5- to 10-fold (relative to the control set or to a Protein Data Bank sample), while quality of covalent geometry, R(work), R(free), electron density and difference density are maintained or improved. Some parts of this correction process are already fully automated; other parts involve manual rebuilding of conformations flagged by the tests as trapped in the wrong local minimum, often altering features of functional significance. The ease and effectiveness of this technique shows that macromolecular crystal structures from either traditional or high-throughput determinations can feasibly reach a new level of excellence in conformational accuracy and reliability.

Crystallography, X-Ray↗
PubMed2005

Development of a new high-throughput screening model for human high density lipoprotein receptor (CLA-1) agonists.

OBJECTIVE: To develop a new high-throughput screening model for human high-density lipoprotein (HDL) receptor (CD36 and LIMPII analogous-1, CLA-1) agonists using CLA-1-expressing insect cells. METHODS: With the total RNA of human hepatoma cells BEL-7402 as template, the complementary DNA (cDNA) of CLA-1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Bac-to-Bac baculovirus expression system was used to express CLA-1 in insect cells. CLA-1 cDNA was cloned downstream of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV) into donor vector pFastBac1 and recombinant pFastBac1-CLA-1 was transformed into E. coli DH10Bac to transpose CLA-1 cDNA to bacmid DNA. Recombinant bacmid-CLA-1 was transfected into Spodoptera frugiperda Sf9 insect cells to produce recombinant baculovirus particles. Recombinant CLA-1 was expressed on the membrane of Sf9 cells infected with the recombinant baculoviruses. A series of parameters of DiI-lipoprotein binding assays of CLA-1-expressing Sf9 cells in 96-well plates were optimized. RESULTS: Western blot analysis and DiI-lipoprotein binding assays confirmed that CLA-1 expressed in insect cells had similar immunoreactivity and ligand binding activity as its native counterpart. A reliable and sensitive in vitro cell-based assay was established to assess the activity of CLA-1 and used to screen agonists from different sample libraries. CONCLUSION: Human HDL receptor CLA-1 was successfully expressed in Sf9 insect cells and a novel high-throughput screening model for CLA-1 agonists was developed. Utilization of this model allows us to identify potent and selective CLA-1 agonists which might possibly be used as therapeutics for atherosclerosis.

Animals↗
PubMed2001

High-throughput proteomics using high-efficiency multiple-capillary liquid chromatography with on-line high-performance ESI FTICR mass spectrometry.

We report on the design and application of a high-efficiency multiple-capillary liquid chromatography (LC) system for high-throughput proteome analysis. The multiple-capillary LC system using commercial LC pumps was operated at a pressure of 10,000 psi to deliver mobile phases through a novel passive feedback valve arrangement that permitted mobile-phase flow path switching and efficient sample introduction. The multiple-capillary LC system uses several serially connected dual-capillary column devices. The dual-capillary column approach eliminates the time delays for column regeneration (or equilibration) since one capillary column was used for a separation while the other was being washed. Several serially connected dual-capillary columns and electrospray ionization (ESI) sources were operated independently and can be used either for "backup" operation or for parallel operation with other mass spectrometers. This high-efficiency multiple-capillary LC system utilizes switching valves for all operations, enabling automated operation. The separation efficiency of the dual-capillary column arrangement, optimal capillary dimensions (column length and packed particle size), capillary regeneration conditions, and mobile-phase compositions and their compatibility with electrospray ionization were investigated. A high magnetic field (11.4 T) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was coupled on-line with this high-efficiency multiple-capillary LC system using an ESI interface. The capillary LC provided a peak capacity of approximately 650, and the 2-D capillary LC-FTICR analysis provided a combined resolving power of > 6 x 10(7) components. For yeast cytosolic tryptic digests > 100,000 polypeptides were detected, and approximately 1,000 proteins could be characterized from a single capillary LC-FTICR analysis using the high mass measurement accuracy (approximately 1 ppm) of FTICR, and likely more if LC retention time information were also exploited for peptide identification.

Chromatography, Liquid↗
PubMed2005

Discovery of a potent and selective 5-ht5A receptor antagonist by high-throughput chemistry.

High-throughput screening of an array of biphenylmethylamines synthesised by high-throughput solid-phase chemistry resulted in the identification of compounds with high-affinity for the 5-ht5A receptor. The structure-activity relationship within this series and further array synthesis led to the identification of the biphenylmethylamine derivative 11, a potent and selective 5-ht5A receptor antagonist.

Animals↗
PubMed2005

Production and characterization of mouse ureteric bud cell-specific rat hybridoma antibodies utilizing subtractive immunization and high-throughput screening.

The highly branched collecting system of the kidney arises developmentally from the ureteric bud (UB) by a process of branching morphogenesis. This process is critical for the normal development of the collecting ducts and pelvis of the kidney, and is tightly controlled by the spatial and temporal expression of numerous proteins. To identify cell proteins that are differentially expressed by the UB relative to those expressed by the highly differentiated collecting ducts of the adult kidney, two mouse cell populations derived from either the early UB or the adult inner medullary collecting duct (IMCD) were used. A subtractive immunization strategy was performed in rats to generate monoclonal antibodies that preferentially reacted with antigens on UB, but not IMCD cells. In addition, the technique of antibody printing, a novel high-throughput antibody screening method for determining the specificities of a large number of monoclonal antibodies, is described. The methodologies outlined in this manuscript have broad applicability as they demonstrate that subtractive immunization can be performed in rats with cells derived from mice. Additionally, the high-throughput screening methods should facilitate the use of subtractive immunization for identifying antibodies that can distinguish differences in proteins expressed in closely related cell types.

Animals↗
PubMed2000

Automatic poisson peak harvesting for high throughput protein identification.

High throughput identification of proteins by peptide mass fingerprinting requires an efficient means of picking peaks from mass spectra. Here, we report the development of a peak harvester to automatically pick monoisotopic peaks from spectra generated on matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometers. The peak harvester uses advanced mathematical morphology and watershed algorithms to first process spectra to stick representations. Subsequently, Poisson modelling is applied to determine which peak in an isotopically resolved group represents the monoisotopic mass of a peptide. We illustrate the features of the peak harvester with mass spectra of standard peptides, digests of gel-separated bovine serum albumin, and with Escherictia coli proteins prepared by two-dimensional polyacrylamide gel electrophoresis. In all cases, the peak harvester proved effective in its ability to pick similar monoisotopic peaks as an experienced human operator, and also proved effective in the identification of monoisotopic masses in cases where isotopic distributions of peptides were overlapping. The peak harvester can be operated in an interactive mode, or can be completely automated and linked through to peptide mass fingerprinting protein identification tools to achieve high throughput automated protein identification.

Animals↗
PubMed2006

A set of UV-inducible autolytic vectors for high throughput screening.

A high throughput screening scheme is often a prerequisite for directed evolution of enzymes or metagenomic analysis of DNA samples. For assaying intracellular enzymes of interest (e.g. when Escherichia coli is used), it requires cell lysis in many cases, chemical or enzymatic, which can be tedious and cost-consuming. In this study, a set of UV-inducible autolytic vectors was constructed to offer a simpler means of cell lysis that is free of additional liquid handling. The SRRz lysis gene cassette from bacteriophage Lambda was cloned downstream of a UV-inducible promoter, the recA promoter or the umuDC promoter, and further inserted into the backbone of pUC18, and transformed into E. coli BL21 cells. The SRRz expression and cell lysis was induced by UV irradiation. For both the recA and umuDC promoters, at 30 degrees C the lysis efficiency was found to be consistent and above 60% as measured using beta-galactosidase as the reporter. However, at 37 degrees C the lysis profiles were found to be erratic. UV lysis in 96-well plates also produced consistent lysis results that were comparable to those obtained by lysozyme treatment, demonstrating the utility of these autolytic vectors in high throughput screening. This set of artificial SRRz autolysis units should be transferable to other vectors. Surprisingly, it was found that the E. coli BL21(DE3) was also partially disrupted under UV irradiation, with a lysis efficiency of 44.5% at 30 degrees C, and 22.5% at 37 degrees C.

Bacteriolysis↗
PubMed2002

A high-throughput denaturing high-performance liquid chromatography method for the identification of variant alleles associated with dihydropyrimidine dehydrogenase deficiency.

Dihydropyrimidine dehydrogenase (DPD) is the initial, rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU). A pharmacogenetic syndrome has been described in which DPD-deficient patients are at risk for toxicity following administration of 5-FU. To date, there are at least 21 previously described mutations and/or polymorphisms that have been associated with DPD deficiency. In this study we describe the development of a highly specific, sensitive, inexpensive, and robust denaturing HPLC (DHPLC) method for rapidly identifying sequence variations (mutations and/or polymorphisms) in the gene (DPYD) that codes for the DPD enzyme. DHPLC conditions were optimized at three temperatures for analysis of the 23 exons of the DPYD gene using 25 amplicons representing the entire coding sequence, including all intron/exon boundaries (splice sites). Resolution of all 25 amplicons at the optimized temperature can be performed in 4.2 h. All 21 previously described sequence variations (mutations and/or polymorphisms) were prepared using site-directed mutagenesis from the wild-type DPYD gene, confirmed by sequence analysis, and subsequently resolved by DHPLC using the optimized conditions. These analyses generated reference chromatogram patterns for all known sequence variations previously encountered in DPD-deficient patients. In order to examine the utility and sensitivity of this approach, samples from patients with known sequence variations in the DPYD gene were analyzed. This DHPLC technique resolved 100% of the known DPYD sequence variations and differentiated between homozygous and heterozygous genotypes. We conclude that this DHPLC method is a highly specific and sensitive technique for rapidly detecting known sequence variations in the DPYD gene. In addition, this approach can be used to identify currently unrecognized unknown sequence variations in the DPYD gene and should be useful in future pharmacogenetic studies examining DPD deficiency.

Alleles↗