Extraction and Solubilization of Proteins for Proteomic Studies Richard M. Leimgruber Preparation of Bacterial Samples for 2-D PAGE Brian Berg Vandahl, Gunna Christiansen, and Svend Birkelund Preparation of Yeast Samples for 2-D PAGE Joakim Norbeck Preparation of Mammalian Tissue Samples for Two-Dimensional Electrophoresis Frank A. Witzmann Differential Detergent Fractionation of Eukaryotic Cells Melinda L. Ramsby and Gregory S. Makowski Serum or Plasma Sample Preparation for Two-Dimensional Gel Electrophoresis Anthony G. Sullivan, Stephen Russell, Henry Brzeski, Richard I. Somiari, and Craig D. Shriver Preparation of Plant Protein Samples for 2-D PAGE David W. M. Leung Laser-Assisted Microdissection in Proteomic Analyses Darrell L. Ellsworth, Stephen Russell, Brenda Deyarmin, Anthony G. Sullivan, Henry Brzeski, Richard I. Somiari, and Craig D. Shriver Purification of Cellular and Organelle Populations by Fluorescence-Activated Cell Sorting for Proteome Analysis William L. Godfrey, Colette J. Rudd, Sujata Iyer, and Diether Recktenwald Purification of Nucleoli From Lymphoma Cells and Solubilization of Nucleolar Proteins for 2-DE Separation Regis Dieckmann, Yohann Coute, Denis Hochstrasser, Jean-Jacques Diaz, and Jean-Charles Sanchez Prefractionation of Complex Protein Mixture for 2-D PAGE Using Reversed-Phase Liquid Chromatography Volker Badock and Albrecht Otto Fractionation of Complex Proteomes by Microscale Solution Isoelectrofocusing Using ZOOM(TM) IEF Fractionators to Improve Protein Profiling Xun Zuo, Ki-Boom Lee, and David W. Speicher Large-Format 2-D Polyacrylamide Gel Electrophoresis Henry Brzeski, Stephen Russell, Anthony G. Sullivan, Richard I. Somiari, and Craig D. Shiver Analysis of Membrane Proteins by Two-Dimensional Gels MichaelFountoulakis 2-D PAGE of High-Molecular-Mass Proteins Masamichi Oh-Ishi and Tadakazu Maeda Using Ultra-Zoom Gels for High-Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis Sjouke Hoving, Hans Voshol, and Jan van Oostrum NEpHGE and pI Strip Proteomic 2-D Gel Electrophoretic Mapping of Lipid-Rich Membranes Steven E. Pfeiffer, Yoshihide Yamaguchi, Cecilia B. Marta, Rashmi Bansal, and Christopher M. Taylor Silver Staining of 2-D Gels Julia Poland, Thierry Rabilloud, and Pranav Sinha Zn2+ Reverse Staining Technique Carlos Fernandez-Patron Multiplexed Proteomics Technology for the Fluorescence Detection of Glycoprotein Levels and Protein Expression Levels Using Pro-Q(R) Emerald and SYPRO(R) Ruby Dyes Birte Schulenberg and Wayne F. Patton Multiplexed Proteomics Technology for the Fluorescence Detection of Phosphorylation and Protein Expression Levels Using Pro-Q(R) Diamond and SYPRO(R) Ruby Dyes Birte Schulenberg, Terrie Goodman, Thomas H. Steinberg, and Wayne F. Patton Sensitive Quantitative Fluorescence Detection of Proteins in Gels Using SYPRO(R) Ruby Protein Gel Stain Birte Schulenberg, Nancy Ahnert, and Wayne F. Patton Rapid, Sensitive Detection of Proteins in Minigels With Fluorescent Dyes: Coomassie Fluor Orange, SYPRO(R) Orange, SYPRO Red, and SYPRO Tangerine Protein Gel Stains Thomas H. Steinberg, Courtenay R. Hart, and Wayne F. Patton Differential In-Gel Electrophoresis in a High-Throughput Environment Richard I. Somiari, Stephen Russell, Stella B. Somiari, Anthony G. Sullivan, Darrell L. Ellsworth, Henry Brzeski, and Craig D. Shriver Statistical Analysis of 2-D Gel Patterns Francoise Seillier-Moiseiwitsch 2-DE Databases on the World Wide Web Christine Hoogland, Khaled Mostaguir, and Ron D. Appel Computer Analysis of 2-D Images Patricia M. Palagi,

The proteomics protocols handbook

The sequence of a genome contains the plans of the possible life of an organism, but implementation of genetic information depends on the functions of the proteins and nucleic acids that it encodes. Many individual proteins of known sequence and structure present challenges to the understanding of their function. In particular, a number of genes responsible for diseases have been identified but their specific functions are unknown. Whole-genome sequencing projects are a major source of proteins of unknown function. Annotation of a genome involves assignment of functions to gene products, in most cases on the basis of amino-acid sequence alone. 3D structure can aid the assignment of function, motivating the challenge of structural genomics projects to make structural information available for novel uncharacterized proteins. Structure-based identification of homologues often succeeds where sequence-alone-based methods fail, because in many cases evolution retains the folding pattern long after sequence similarity becomes undetectable. Nevertheless, prediction of protein function from sequence and structure is a difficult problem, because homologous proteins often have different functions. Many methods of function prediction rely on identifying similarity in sequence and/or structure between a protein of unknown function and one or more well-understood proteins. Alternative methods include inferring conservation patterns in members of a functionally uncharacterized family for which many sequences and structures are known. However, these inferences are tenuous. Such methods provide reasonable guesses at function, but are far from foolproof. It is therefore fortunate that the development of whole-organism approaches and comparative genomics permits other approaches to function prediction when the data are available. These include the use of protein-protein interaction patterns, and correlations between occurrences of related proteins in different organisms, as indicators of functional properties. Even if it is possible to ascribe a particular function to a gene product, the protein may have multiple functions. A fundamental problem is that function is in many cases an ill-defined concept. In this article we review the state of the art in function prediction and describe some of the underlying difficulties and successes.

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Prediction of protein function from protein sequence and structure.

By far the largest proportion of the Earth's biosphere is comprised of organisms that thrive in cold environments (psychrophiles). Their ability to proliferate in the cold is predicated on a capacity to synthesize cold-adapted enzymes. These enzymes have evolved a range of structural features that confer a high level of flexibility compared to thermostable homologs. High flexibility, particularly around the active site, is translated into low-activation enthalpy, low-substrate affinity, and high specific activity at low temperatures. High flexibility is also accompanied by a trade-off in stability, resulting in heat lability and, in the few cases studied, cold lability. This review addresses the structure, function, and stability of cold-adapted enzymes, highlighting the challenges for immediate and future consideration. Because of the unique properties of cold-adapted enzymes, they are not only an important focus in extremophile biology, but also represent a valuable model for fundamental researc...

Cold-adapted enzymes.

Profound changes are occurring in the strategies that biotechnology-based industries are deploying in the search for exploitable biology and to discover new products and develop new or improved processes. The advances that have been made in the past decade in areas such as combinatorial chemistry, combinatorial biosynthesis, metabolic pathway engineering, gene shuffling, and directed evolution of proteins have caused some companies to consider withdrawing from natural product screening. In this review we examine the paradigm shift from traditional biology to bioinformatics that is revolutionizing exploitable biology. We conclude that the reinvigorated means of detecting novel organisms, novel chemical structures, and novel biocatalytic activities will ensure that natural products will continue to be a primary resource for biotechnology. The paradigm shift has been driven by a convergence of complementary technologies, exemplified by DNA sequencing and amplification, genome sequencing and annotation, proteome analysis, and phenotypic inventorying, resulting in the establishment of huge databases that can be mined in order to generate useful knowledge such as the identity and characterization of organisms and the identity of biotechnology targets. Concurrently there have been major advances in understanding the extent of microbial diversity, how uncultured organisms might be grown, and how expression of the metabolic potential of microorganisms can be maximized. The integration of information from complementary databases presents a significant challenge. Such integration should facilitate answers to complex questions involving sequence, biochemical, physiological, taxonomic, and ecological information of the sort posed in exploitable biology. The paradigm shift which we discuss is not absolute in the sense that it will replace established microbiology; rather, it reinforces our view that innovative microbiology is essential for releasing the potential of microbial diversity for biotechnology penetration throughout industry. Various of these issues are considered with reference to deep-sea microbiology and biotechnology.

Search and Discovery Strategies for Biotechnology: the Paradigm Shift

Comparative genomics shows that a substantial fraction of the genes in sequenced genomes encodes ‘conserved hypothetical’ proteins, i.e. those that are found in organisms from several phylogenetic lineages but have not been functionally characterized. Here, we briefly discuss recent progress in functional characterization of prokaryotic ‘conserved hypothetical’ proteins and the possible criteria for prioritizing targets for experimental study. Based on these criteria, the chief one being wide phyletic spread, we offer two ‘top 10’ lists of highly attractive targets. The first list consists of proteins for which biochemical activity could be predicted with reasonable confidence but the biological function was predicted only in general terms, if at all (‘known unknowns’). The second list includes proteins for which there is no prediction of biochemical activity, even if, for some, general biological clues exist (‘unknown unknowns’). The experimental characterization of these and other ‘conserved hypothetical’ proteins is expected to reveal new, crucial aspects of microbial biology and could also lead to better functional prediction for medically relevant human homologs.

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‘Conserved hypothetical’ proteins: prioritization of targets for experimental study

Almost half of the entire set of predicted genomic products from Methanococcus jannaschii are classified as functionally unknown hypothetical proteins. We present a structure-based identification of the biochemical function of a protein with an as yet unknown function from a M. jannaschii gene, Mj0226. The crystal structure of Mj0226 protein determined at 2.2 A resolution reveals that the protein is a homodimer and each monomer folds into an elongated α/β structure of a new fold family. Comparisons of Mj0226 protein with protein structures in the database, however, indicate that one part of the protein is homologous to some of the nucleotide-binding proteins. Biochemical analysis shows that Mj0226 protein is a novel nucleotide triphosphatase that can efficiently hydrolyze nonstandard nucleotides such as XTP to XMP or ITP to IMP, but not the standard nucleotides, in the presence of Mg2+ or Mn2+ ions.

Ji Hyung Chung

Papers

Structure-based identification of a novel NTPase from Methanococcus jannaschii