Membrane Proteomics

This book collects together in one volume the diverse practical approaches to analyzing membrane proteins. This is an ideal guide for investigators wishing to apply state-of-the-art membrane proteomic methodologies in their own research programs.

The membranes surrounding cells and organelles constitute their interface with the local environment. The functions of membrane proteins include cell/cell and cell/extracellular matrix recognition, the reception and transduction of extracellular signals, and the tra- port of proteins, solutes and water molecules. Abnormal membrane protein expression has profound biological effects and may, for example, underlie phenotypic and functional differences between normal and tumour cells. Moreover the accessibility, particularly of plasma proteins traversing the plasma membrane of cells, makes them of particular ut- ity to the therapeutic intervention in disease. Indeed, it is estimated that of all currently licensed pharmaceuticals, approximately 70% target proteins resident in the plasma m- brane. In theory, unbiased technologies such as proteomics have the power to de?ne patterns of membrane protein expression characteristic of distinct states of cellular development, differentiation or disease, and thereby identify novel markers of, or targets for intervention in, disease. However, although about 25% of open reading frames in fully sequenced genomes are estimated to encode integral membrane proteins, global analysis of membrane protein expression has proved problematic. Membrane protein analysis poses unique challenges at the level of extraction, solubilization, and separation in particular, and to a lesser extent of identi?cation and quantitation. These challenges have, however, fostered creativity, in- vation, and technical advances, many of which are brought together in Membrane P- teomics.

Klappentext

With the great cellular and therapeutic importance of plasma membrane proteins, unbiased technologies such as proteomics become even more vital as they have the power to define patterns of membrane protein expression characteristic of distinct states of cellular development, differentiation or disease, and thereby identify novel markers of, or targets for intervention in, disease. In Membrane Proteomics: Methods and Protocols, leading experts in the field compile a laboratory bench resource which provides a comprehensive toolkit of proven methods. The volume delves into various modifications to standard two-dimensional gel electrophoresis protocols as well as liquid chromatographic methods, protocols for the enrichment of diverse classes of plasma membrane proteins, in silico approaches, and cutting-edge techniques for quantitative membrane protein profiling. Written in the highly successful Methods in Molecular Biology™ series format, chapters include brief 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.

Comprehensive and easy-to-use, Membrane Proteomics: Methods and Protocols is an ideal guide for investigators wishing to apply state of the art membrane proteomic methodologies in their own research programs.

Inhalt
In Silico Methods for Prediction of Membrane Protein Hydrophobicity and Topology.- Online Tools for Predicting Integral Membrane Proteins.- In Silico Identification of Novel G Protein Coupled Receptors.- Transcriptome-Based Identification of Candidate Membrane Proteins.- Extraction and Purification of Membrane Proteins.- Separation of Thylakoid Membrane Proteins by Sucrose Gradient Ultracentrifugation or Blue Native-SDS-PAGE Two-Dimensional Electrophoresis.- Extraction of Yeast Mitochondrial Membrane Proteins by Solubilization and Detergent/Polymer Aqueous Two-Phase Partitioning.- 16-BAC/SDS-PAGE Analysis of Membrane Proteins of Yeast Mitochondria Purified by Free Flow Electrophoresis.- Sequential Detergent Extraction Prior to Mass Spectrometry Analysis.- Enrichment of Brain Plasma Membranes by Affinity Two-Phase Partitioning.- Protocol to Enrich and Analyze Plasma Membrane Proteins.- Proteomic Analysis of the Lymphocyte Plasma Membrane Using Cell Surface Biotinylation and Solution-Phase Isoelectric Focusing.- Identification of Target Membrane Proteins as Detected by Phage Antibodies.- Membrane Protease Degradomics: Proteomic Identification and Quantification of Cell Surface Protease Substrates.- Purification of Basolateral Integral Membrane Proteins by Cationic Colloidal Silica-Based Apical Membrane Subtraction.- Moving Closer to the Lipid Raft Proteome Using Quantitative Proteomics.- Use of Sequential Chemical Extractions to Purify Nuclear Membrane Proteins for Proteomics Identification.- Isolation of Extracellular Membranous Vesicles for Proteomic Analysis.- Separation of Membrane Proteins.- Enrichment of Human Platelet Membranes for Proteomic Analysis.- Detergents and Chaotropes for Protein Solubilization Before Two-Dimensional Electrophoresis.- Two-Dimensional Separation of Membrane Proteins by 16-BAC-SDS-PAGE.- Identification and Quantification of Membrane Proteins.- MudPIT Analysis: Application to Human Heart Tissue.- Liquid Chromatography MALDI MS/MS for Membrane Proteome Analysis.- Cysteinyl-Tagging of Integral Membrane Proteins for Proteomic Analysis Using Liquid Chromatography-Tandem Mass Spectrometry.- Quantitative Proteomics Analysis of Pancreatic Zymogen Granule Membrane Proteins.