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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.
Table des matières
Two-Dimensional Electrophoresis: An Overview.- Solubilization of Proteins in 2DE: An Outline.- Selection of pH Ranges in 2DE.- Difficult Proteins.- Organelle Proteomics.- Applications of Chemical Tagging Approaches in Combination with 2DE and Mass Spectrometry.- Immunoblotting 2DE Membranes.- Troubleshooting Image Analysis in 2DE.- Analysis of Bacterial Proteins by 2DE.- Proteomic Analysis of Caenorhabditis elegans.- Protein Extraction for 2DE.- Analysis of Proteins from Marine Molluscs.- Preparation and Analysis of Plant and Plastid Proteomes by 2DE..- High-Resolution 2DE.- Blue Native-Gel Electrophoresis Proteomics.- 2DE for Proteome Analysis of Human Metaphase Chromosomes.- Microsomal Proteomics.- Prefractionation Using Microscale Solution IEF.- Diagonal Electrophoresis for Detection of Protein Disulphide Bridges.- High-Resolution Large-Gel 2DE.- Silver Staining of Proteins in 2DE Gels.- Detection of 4-Hydroxy-2-Nonenal- and 3-Nitrotyrosine-Modified Proteins Using a Proteomics Approach.- Proteomic Detection of Oxidized and Reduced Thiol Proteins in Cultured Cells.- Detection of Ubiquitination in 2DE.- Phosphoproteome Analysis by In-Gel Isoelectric Focusing and Tandem Mass Spectrometry.- Detection of Protein Glutathionylation.- Activity-Based Protein Profiling of Protein Tyrosine Phosphatases.- Active Protease Mapping in 2DE Gels.- Two-Dimensional Difference Gel Electrophoresis.- Protein Expression Profiling.- C-Terminal Sequence Analysis of 2DE-Separated Proteins.- Shotgun Protein Analysis by Liquid Chromatography-Tandem Mass Spectrometry.- de Novo Sequence Analysis of N-Terminal Sulfonated Peptides After in-Gel Guanidination.- Tryptic Digestion of In-Gel Proteins for Mass Spectrometry Analysis.- Database Interrogation Algorithms for Identification of Proteins in Proteomic Separations.- Creating 2DE Databases for the World Wide Web.
Résumé
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.
