Abstract
Tal V. Murthy Caliper Life Sciences Hopkinton, MA
Laboratory Automation and High-Throughput Chemistry
Integrating High-Throughput Genetic Interaction Mapping and High-Content Screening to Explore Yeast Spindle Morphogenesis
In this study, F. J. Vizeacoumar et al. from the University of Toronto describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. The authors measure numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. They focus on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, and the cell cycle regulatory networks, mitotic exit network, and fourteen early anaphase release. They also dissect the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although the authors focus on spindle disassembly in a proof-of-principle study, their integrated HCS—SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions (J. Cell Biol.
Rapid, Semiautomated, and Inexpensive Radioimmunoassay of cAMP: Application in G Protein-Coupled Receptor-Mediated Adenylate Cyclase Assays
Cyclic adenosine monophosphate is an important signal transduction second messenger that is commonly used as a functional mirror on the actions of G protein-coupled receptors that can activate or inhibit adenylate cyclases. A radioimmunoassay for cyclic adenosine monophosphate with femtomole sensitivity was first reported by Steiner more than 30 years ago, and there have been several subsequent modifications that have improved this assay in various ways. Here, the authors describe additional improvement to existing methods that markedly improve speed and reduce cost without sacrificing sensitivity, and is also adaptable to analysis of cyclic guanosine monophosphate. The primary antibody is coupled directly to magnetic beads that are separated from unbound marker using filtration on microplates. This eliminates the need for a secondary antibody, and markedly increases throughput. In addition, a simple, reproducible, and inexpensive method to make the radiomarker used for this assay is reported. Although still requiring the use of radioactivity, the resulting method retains a high degree of accuracy and precision, and is suitable for low-cost high-throughput screening. Use of aspects of this method can also improve throughput in other radioimmunoassays (J. Neurosci. Methods.
Sensitive and Convenient Yeast Reporter Assay for High-Throughput Analysis by Using a Secretory Luciferase from Cypridina Noctiluca
Novel alternatives for routine reporter assays offer options to researchers during design of experiments. One such alternative to beta-galactosidase reporter assay is reported by authors Y. Tochigi et al. in this study. The yeast reporter assay has been widely used in various applications such as detection of endocrine disruptors and analysis of protein-protein interactions by the yeast two-hybrid system. The molecular characteristics of the reporter enzyme are critical determinants for this assay.
The authors establish a novel yeast reporter assay using a secretory luciferase, Cypridina noctiluca luciferase (CLuc) as an alternative to the conventional beta-galactosidase. The CLuc reporter assay in yeast is more sensitive and convenient than the conventional assay. The yeast high-throughput reporter assay is established with a laboratory automation system, and the transcriptional activity of hundreds of yeast promoter fragments is comprehensively determined. Results indicate that the yeast CLuc reporter assay is a promising tool for large-scale and sensitive analysis in the development of new drugs and in various fields of biotechnology research (Anal. Chem.
Microfluidic Chip Technology and Micro Reactor Technology
”Click“ Chemistry-Based Surface Modification of Poly (dimethylsiloxane) for Protein Separation in a Microfluidic Chip
Z. Zhang et al. present a surface modification for protein separation in a microfluidic chip. ”Click“ chemistry-based surface modification strategy was developed for polydime-thylsiloxane (PDMS) microchips to enhance separation performance for both amino acids and proteins. Alkyne-polyethylene glycol (PEG) is synthesized by a conventional procedure and then ”click“ grafted to azido-PDMS. FTIR absorption by attenuated total reflection and contact angle measurements proved efficient grafting of alkyne-PEG onto PDMS surface. Manifest electroosmotic flow (EOF) regulation and stability of PEG-functionalized PDMS microchips are illustrated via EOF measurements and protein adsorption investigations. The stability of nonspecific protein adsorption resistance property is investigated up to 30 days. Separation of fluorescence-labeled amino acids and proteins is further demonstrated with high repeatability and reproducibility. Comparison of protein separation using PDMS microchips before and after surface modification suggests greatly improved electrophoretic performance of the PEG-functionalized PDMS microchips. The authors expect the ”click“ chemistry-based surface modification method to have wide applications in microseparation of proteins with long-term surface stability (Electrophoresis.
Microfluidic Serial Dilution Cell-Based Assay for Analyzing Drug-Dose Response over a Wide Concentration Range
In this article, S. Sugiura et al. report a perfusion culture microchamber array chip with a serial dilution microfluidic network for analyzing drug-dose response over a concentration range spanning six orders of magnitude, which is required for practical drug discovery applications. The microchamber array chip is equipped with a pressure-driven interface, in which medium and drug solution are added with a micropipet and delivered into the microfluidic network by pneumatic pressure. The authors demonstrate that the microchamber array chip can be used to estimate the 50% growth inhibitory concentration using the model anticancer drug paclitaxel and the model cancer cell line HeLa. The results obtained using the microchamber array chip are consistent with those obtained by a conventional assay using microplates. The microchamber array chip, with its simple interface and well-designed microfluidic network, has potential as an efficient platform for high-throughput dose response assays in drug discovery applications (Anal. Chem.
Accessing Protein Methyltransferase and Demethylase Enzymology Using Microfluidic Capillary Electrophoresis
T. J. Wigle et al. from Janzen's lab at University of North Carolina reports a quantitative microfluidic capillary electrophoresis assay to study the mechanism of action of enzymes. This microfluidic assay is successfully used to investigate the inhibition of a methyltransferase by small molecules. The discovery of small molecules targeting the >80 enzymes that add (methyltransferases) or remove (demethylases) methyl marks from lysine and arginine residues, most notably present in histone tails, may yield unprecedented chemotherapeutic agents and facilitate regenerative medicine. To better enable chemical exploration of these proteins, a highly quantitative microfluidic capillary electrophoresis assay to enable full mechanistic studies of these enzymes and the kinetics of their inhibition is developed. This technology separates small biomolecules, that is, peptides, based on their charge-to-mass ratio. Methylation, however, does not alter the charge of peptide substrates. To overcome this limitation, a methylation-sensitive endoproteinase strategy to separate methylated from unmethylated peptides is used. The assay is validated on a lysine methyltransferase (G9a) and a lysine demethylase and is used to investigate the inhibition of G9a by small molecules (Chem. Biol.
High-Throughput Analytics
Systems Biology Approaches and Tools for Analysis of Interactomes and Multitarget Drugs
Systems biology is essentially a proteomic and epigenetic exercise because the relatively condensed information of genomes unfolds on the level of proteins. The flexibility of cellular architectures is not only mediated by a dazzling number of proteinaceous species but moreover by the kinetics of their molecular changes: The time scales of posttranslational modifications range from milliseconds to years.
The genetic framework of an organism only provides the blue print of protein embodiments that are constantly shaped by external input. Posttranslational modifications of proteins represent the scope and velocity of these inputs and fulfill the requirements of integration of external spatiotemporal signal transduction inside an organism. The optimization of biochemical networks for this type of information processing and storage results in chemically extremely fine-tuned molecular entities. The huge dynamic range of concentrations, the chemical diversity, and the necessity of synchronization of complex protein expression patterns pose the major challenge of systemic analysis of biological models.
Many of the key reactions in living systems are essentially based on interactions of moderate affinities and moderate selectivity. This principle is responsible for the enormous flexibility and redundancy of cellular circuitries. In complex disorders such as cancer or neurodegenerative diseases, which initially appear to be rooted in relatively subtle dysfunctions of multimodal physiologic pathways, drug discovery programs based on the concept of high-affinity/high-specificity compounds (“one target, one disease”), which has been dominating the pharmaceutical industry for a long time, increasingly turn out to be unsuccessful. Despite improvements in rational drug design and high-throughput screening methods, the number of novel, single-target drugs fell much behind expectations during the past decade, and the treatment of “complex diseases” remains a most pressing medical need.
Currently, a change of paradigm can be observed with regard to a new interest in agents that modulate multiple targets simultaneously, essentially “dirty drugs.” Targeting cellular function as a system rather than on the level of the single target significantly increases the size of the drugable proteome and is expected to introduce novel classes of multitarget drugs with fewer adverse effects and toxicity. Multiple target approaches have recently been used to design medications against atherosclerosis, cancer, depression, psychosis, and neurodegenerative diseases.
A focused approach toward “systemic” drugs will certainly require the development of novel computational and mathematical concepts for appropriate modeling of complex data. But the key is the extraction of relevant molecular information from biological systems by implementing rigid statistical procedures to differential proteomic analytics (Schrattenholz, A. et al. Methods Mol. Biol.
Automation Systems
Robust, High-Throughput Solution for Blood-Group Genotyping
With the concomitant increase of blood transfusions and safety rules, there is a growing need to integrate high-throughput and multiparametric assays within blood qualification centers. Using a robust and automated solution, G. C. LeGoff et al. describe a new method for extended blood-group genotyping (HiFi-Blood 96), bringing together the throughput possibilities of complete automation and the microarray multiplexed analysis potential. Their approach provides a useful resource for upgrading blood qualification center facilities.
A set of six single-nucleotide polymorphisms (SNPs) associated with clinically important blood-group antigens (Kell, Kidd, Duffy, and MNS systems) are selected and the corresponding genotyping assays developed. A panel of 293 blood samples is used to validate the approach. The resulting genotypes are compared with phenotypes previously determined by standard serologic techniques, and excellent correlations are found for five SNPs out of six. For the Kell, Kidd, Duffy, and MNS3/MNS4 systems, high-matching percentages of 100%, 98.9%, 97.7%, and 97.4% are obtained, respectively, whereas a concordance percentage of 83.3% only is attained for the MNS1/MNS2 polymorphism (Anal. Chem.
Automated Quantitative Live-Cell Fluorescence Microscopy
In this review, M. Fero and K. Pogliano present an overview of automated image analysis and discuss some of the applications and challenges. Advances in microscopy automation and image analysis have given biologists the tools to attempt large-scale systems-level experiments on biological systems using microscope image readout. Fluorescence microscopy has become a standard tool for assaying gene function in RNAi knockdown screens and protein localization studies in eukaryotic systems. Similar high-throughput studies can be attempted in prokaryotes, although the difficulties surrounding work at the diffraction limit pose challenges, and targeting essential genes in a high-throughput way can be difficult.
The authors discuss efforts to make live-cell fluorescent microscopy-based experiments using genetically encoded fluorescent reporters an automated, high-throughput, and quantitative endeavor amenable to systems-level experiments in bacteria. They emphasize a quantitative data reduction approach, using simulation to help develop biologically relevant cell measurements that completely characterize the cell image. An example of how this type of data can be directly exploited by statistical learning algorithms to discover functional pathways is presented (Cold Spring Harb. Perspect. Biol.
Significantly Improved Precision of Cell Migration Analysis in Time-Lapse Video Microscopy Through Use of a Fully Automated Tracking System
J. Huth et al. report an attempt to improve the study of cell migration analysis. Their experimental results demonstrate that the use of automated tracking system yields superior precision compared with manual methods.
Cell motility is a critical parameter in many physiological and pathophysiological processes. In time-lapse video microscopy, manual cell tracking remains the most common method of analyzing migratory behavior of cell populations. In addition to being labor intensive, this method is susceptible to user-dependent errors regarding the selection of “representative” subsets of cells and manual determination of precise cell positions.
The authors quantitatively analyzed these error sources, demonstrating that manual cell tracking of pancreatic cancer cells lead to miscalculation of migration rates of up to 410%. To provide for objective measurements of cell migration rates, the authors used multitarget tracking technologies commonly used in radar applications to develop fully automated cell identification and tracking system suitable for high-throughput screening of video sequences of unstained living cells.
In conclusion, the authors demonstrate that their automatic multitarget tracking system identifies cell objects, follows individual cells, and computes migration rates with high precision, clearly outperforming manual procedures (Huth, J. et al. BMC Cell Biol.
Advances in High-Throughput Protein Purification for Structure Determination
Cell-Free Protein Synthesis Technology in Nuclear Magnetic Resonance (NMR) High-Throughput Structure Determination
This chapter by S. Makino et al. describes the current implementation of the cell-free translation platform developed at the Center for Eukaryotic Structural Genomics (CESG) and practical aspects of the production of stable isotope-labeled eukaryotic proteins for NMR structure determination.
Protocols are reported for the use of wheat germ cell-free translation in small-scale screening for the level of total protein expression, the solubility of the expressed protein, and the success in purification as predictive indicators of the likelihood that a protein may be obtained in sufficient quantity and quality to initiate structural studies. In most circumstances, the small-scale reactions also produce sufficient protein to permit bioanalytical and functional characterizations. The protocols incorporate the use of robots specialized for small-scale cell-free translation, large-scale protein production, and automated purification of soluble, His(6)-tagged proteins. The integration of isotopically labeled proteins into the sequence of experiments required for NMR structure determination is outlined, and the additional protocols for production of integral membrane proteins in the presence of either detergents or unilamellar liposomes are presented (Methods Mol. Biol.
The Scottish Structural Proteomics Facility: Targets, Methods, and Outputs
Over the last five decades, the progress in obtaining three-dimensional protein structures was relatively slow. Several facilities across different countries initiated high-throughput attempts to obtain three-dimensional structures. These structure repositories allow researchers to use the structural information in their respective individual research. One such facility that was attempting the high-throughput structure determination is the Scottish Structural Proteomics Facility. The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high-throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are described in this report.
This report reflects on the value of automation and the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. M. Oke et al. from University of St Andrews, UK, report that in the 5-year period they published 10 peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded their ability to analyze and publish each new finding. By reporting the experimental details and depositing the structures, they hope to maximize the impact of the project by allowing others to follow up the relevant biology (J. Struct. Funct. Genomics.
High-Throughput Crystallography for Structural Genomics
A. Joachimiak from the Midwest Center for Structural Genomics at Argonne National Laboratory discusses the advancements in high-throughput protein crystallography and highlights the tools that play an important role in making multiple structure determinations possible. Protein X-ray crystallography recently celebrated its 50th anniversary. The structures of myoglobin and hemoglobin determined by Kendrew and Perutz provide the first glimpses into the complex protein architecture and chemistry. Since then, the field of structural molecular biology has experienced extraordinary progress and now more than 55,000 protein structures have been deposited into the Protein Data Bank.
In the past decade many advances in macromolecular crystallography have been driven by worldwide structural genomics efforts. This was made possible because of third-generation synchrotron sources, structure-phasing approaches using anomalous signal, and cryo-crystallography. Complementary progress in molecular biology, proteomics, hardware, and software for crystallographic data collection; structure determination and refinement; computer science; databases; robotics; and automation improved and accelerated many processes. These advancements provide the robust foundation for structural molecular biology and assure strong contribution to science in the future. This report focuses mainly on reviewing structural genomics high-throughput X-ray crystallography technologies and their impact (Curr. Opin. Struct. Biol.
The High-Throughput Protein Sample Production Platform of the Northeast Structural Genomics Consortium
This publication by R. Xiao et al. describes their experience on high-throughput protein-production platform for structural genomics efforts from the Northeast Structural Genomics Consortium (NESG), NJ. They describe the core protein-production platform of the NESG and outline the strategies used for producing high-quality protein samples. The platform is centered on the cloning, expression, and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems. The 6X-His tag allows for similar purification procedures for most targets and implementation of high-throughput (HTP) parallel methods.
In most cases, the 6X-His-tagged proteins are sufficiently purified (>97% homogeneity) using a HTP two-step purification protocol for most structural studies. Using this platform, the open-reading frames of over 16,000 different targeted proteins (or domains) have been cloned as >26,000 constructs. Over the past 10 years, more than 16,000 of these expressed protein and more than 4400 proteins (or domains) have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html). Using these samples, the NESG has deposited more than 900 new protein structures to the Protein Data Bank.
The methods described in this report are effective in producing eukaryotic and prokaryotic protein samples in E. coli. This article summarizes some of the updates made to the protein production pipeline in the last 5 years, corresponding to phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative project. The NESG protein production platform is suitable for implementation in a large individual laboratory or by a small group of collaborating investigators. These advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are of broad value to the structural biology, functional proteomics, and structural genomics communities (J. Struct. Biol.