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With the completion of sequencing projects and the advancement of a- lytical tools for protein identification, proteomics—the study of the expressed part of the genome—has become a major region of the burgeoning field of functional genomics. High-resolution 2-D gels can reveal virtually all p- teins present in a cell or tissue at any given time, including posttranslationally modified proteins. Changes in the expression and structure of most cellular proteins caused by differentiation or external stimuli can be displayed and eventually identified using 2-D protein gels. 2-D Proteome Analysis Protocols covers all aspects of the use of 2-D protein electrophoresis for the analysis of biological problems. The contri- tors include many of the leaders in the fields of biochemistry and analytical chemistry who were instrumental in the development of high-resolution 2-D gels, immobilized pH gradients, computer analysis, and mass spectromet- based protein identification methodologies. This book is intended as a benchtop manual and guide both for novices to 2-D gels and for those aficionados who wish to try the newer techniques. Any group using protein biochemistry—especially in the fields of molecular biology, biochemistry, microbiology, and cell biology—should find this book eminently useful. 2-D Proteome Analysis Protocols takes the researcher through the c- plete process of working with 2-D protein gels from making the protein - tract to finally identifying the proteins of interest. It includes protocols for generating 2-D protein extracts from most of the standard model organisms, including bacteria, yeast, nematode, Drosophila, plants, mouse, and human.
Hands-on researchers describe in step-by-step detail 73 proven laboratory methods and bioinformatics tools essential for analysis of the proteome. These cutting-edge techniques address such important tasks as sample preparation, 2D-PAGE, gel staining, mass spectrometry, and post-translational modification. There are also readily reproducible methods for protein expression profiling, identifying protein-protein interactions, and protein chip technology, as well as a range of newly developed methodologies for determining the structure and function of a protein. The bioinformatics tools include those for analyzing 2D-GEL patterns, protein modeling, and protein identification. All laboratory-based protocols follow the successful Methods in Molecular BiologyTM series format, each offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
The human genome and other large-scale genome sequencing projects have inevitably led to a focus on the proteins encoded by genes. The field of proteomics has grown enormously as a result and a number of high-throughput technologies have now been developed allowing discovery-led investigations of protein populations rather than more traditional hypothesis-led studies on single proteins. These high-throughput techno- gies include gene and protein microarrays, the yeast two-hybrid system, and various mass spectrometry methodologies. However, despite developments and improvements in these technologies, two-dimensional electrophoresis (2DE) remains one of the most widely used approaches. This technique was revolutionised by the development of immobilised pH gradient strips which are now commercially available. This has made possible highly reproducible separations of matched samples. Developments in staining, mass spectr- etry, and bioinformatics supported these developments and have led to a measure of standardisation in design, execution, and analysis of proteomics experiments. This book began life as a proposed update of the excellent volume 2DE Protocols edited by Andrew Link of the University of Washington at Seattle. However, we re- ised that 2DE has undergone major development in aspects of its technology in recent years and we were anxious to reflect these in the present volume. We are also conscious that many researchers have now begun to apply proteomics methodologies to a growing range of biological material and we were anxious to include contributions to reflect the challenges posed in sample preparation in less widely used organisms.
This book explores the current status of proteomics, an exciting new discipline, which is less than 10 years old. This new field has rapidly grown into a major commercial and research enterprise with great prospects for dramatically advancing our knowledge of basic biological and disease processes. The contributors to this book are an international panel of proteomics experts, who review and discuss the current status of specific technologies and approaches. Proteomics represents an exciting new way to pursue biological and biomedical science at an unprecedented pace. Proteomics takes a broad, comprehensive, systematic approach to understanding biology that is generally unbiased and not dependent upon existing knowledge. The major components of proteomics from basic discovery using a range of alternative analytical methods to discovery validation and use for clinical applications are discussed. State-of-the-art protein profiling methods include high resolution two-dimensional gels, two-dimensional differential in-gel electrophoresis, LC-MS and LC-MS/MS using accurate mass tags, and protein identifications of proteins from gels using mass spectrometry methods are discussed in depth. Other chapters describe comprehensive characterization of proteomes using electrophoretic prefractionation and analyses of sub-proteomes based on specific posttranslational modifications including the phospho-proteome, the glyco-proteome, and nitrated proteins. These conventional proteome analysis chapters are complemented by discussion of emerging technologies and approaches such as affinity based biosensor proteomics as well as the use of protein microarrays, microfluidics and nanotechnology. Strategies for improving throughput by automation are also discussed. Additional chapters address the application of current proteome techniques to clinical problems and the availability of protein expression library resources for proteome studies.· Authored by international experts in the field · Covers a wide range of topics including 2-D gels, global proteomics using accurate mass tags, global proteomics using electrophoretic prefractionation, microfluidics, and nanotechnology· Includes state-of-the-art protein profiling methods, and emerging technologies
With the end of the Human Genome Project in sight, the next important step is to determine the function of genes. Proteome Research is an important approach to this study and is the first book to comprehensively cover the application of two-dimensional electrophoresis, the central methodology in proteome research. The state-of-the-art is described in detail and the available detection methods are extensively covered. Sufficient detail is given to allow readers to apply these technologies to their own particular requirements.
Protein analysis is increasingly becoming a cornerstone in deciphering the molecular mechanisms of life. Proteomics, the large-scale and high-sensitivity analysis of proteins, is already pivotal to the new life sciences such as Systems Biology and Systems Medicine. Proteomics, however, relies heavily on the past and future advances of protein purification and analysis methods. DIGE, being able to quantify proteins in their intact form, is one of a few methods that can facilitate this type of analysis and still provide the protein isoforms in an MS-compatible state for further identification and characterization with high analytical sensitivity. Differential Gel Electrophoresis: Methods and Protocols introduces the concept of DIGE and its advantages in quantitative protein analysis. It provides detailed protocols and important notes on the practical aspects of DIGE with both generic and specific applications in the various areas of Quantitative Proteomics. Divided into four concise sections, this detailed volume opens with the basics of DIGE, the technique and its practical details with a focus on the planning of a DIGE experiment and its data analysis. The next section introduces various DIGE methods from those employed by scientists world-wide to more novel methods, providing a glance at what is on the horizon in the DIGE world. The volume closes with an overview of the wide range of DIGE applications from Clinical Proteomics to Animal, Plant, and Microbial Proteomics applications. Written in the highly successful Methods in Molecular BiologyTM series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and accessible, Differential Gel Electrophoresis: Methods and Protocols can be used by novices with some background in biochemistry or molecular biology as well as by experts in Proteomics who would like to deepen their understanding of DIGE and its employment in many hyphenations and application areas. With its many protocols, applications, and methodological variants, it is also a unique reference for all who seek fundamental details on the working principle of DIGE and ideas for possible future uses of DIGE in novel analytical approaches.
In this, the post-genomic age, our knowledge of biological systems continues to expand and progress. As the research becomes more focused, so too does the data. Genomic research progresses to proteomics and brings us to a deeper understanding of the behavior and function of protein clusters. And now proteomics gives way to neuroproteomics as we beg
Prostate cancer is the second leading cancer in men in Western society. A major concern, and an area of intensive research, involves understanding why certain prostate cancers remain localized or indolent, whereas others become aggressive and metastasize. The differences between these cancer types have profound implications for patients and physicians. Indolent d- ease, which grows very slowly, generally does not cause any problems to the patient, whereas aggressive disease requires immediate treatment, the earlier the better. At present, there are no markers that discriminate between these two entities, thus causing a dilemma for the management of patients who have recently been diagnosed. The aim of Prostate Cancer Methods and Protocols is to explore cutting-edge molecular methods that may have the potential to reveal markers of disease for use in more accurate diagnoses of prostate c- cer and, consequently, to lead to new treatment strategies. This book provides a comprehensive collection of both in vitro and in vivo step-by-step protocols currently used by leaders in prostate cancer research, advice on approaches that can be used in the study of prostate cancer, as well as reviews covering areas less amenable to laboratory research, such as environmental factors in prostate cancer, to provide the reader with an overview of the prostate cancer research field as it currently stands.
Simpson (food science and agricultural chemistry, McGill U., Canada) brings together academics and industry professionals working in food biochemistry, processing, and safety around the world for this 45-chapter textbook aimed at food scientists, researchers and technologists in the food industry, and faculty and students in food science, technology, and engineering. It combines the areas of food biochemistry and food processing to help them rationalize and develop more effective strategies to produce and preserve food. It covers the essential principles of food biochemistry, enzymology, and food processing, then the biochemistry of meat, poultry, seafoods, milk, fruits, vegetables, cereals, and fermented foods, and food microbiology and safety. Along with updates to several chapters, this edition has been revised to incorporate safety considerations and the chemical changes induced by processing in the biomolecules of food in each chapter. It includes a new section on health and functional foods and 10 new chapters on topics like thermally and minimally processed foods, separation technology, and allergens.
This laboratory guide for successful electrophoretic separations is divided into two parts to provide readers with a thorough presentation of the fundamentals followed by a detailed description of the most common methods currently in use. This fourth edition retains the successful concept of its predecessors, yet features a brand-new layout, and is further enhanced by a section on difference gel electrophoresis, while the chapter on proteome analysis is practically all new and considerably extended, plus there are now around 10 % new literature references.