Publisher: Humana Press
Number Of Pages: 440
Publication Date: 2008-02-19
ISBN-10 / ASIN: 1588297799
ISBN-13 / EAN: 9781588297792
Binding: Hardcover
This book starts with a chapter by Dr. Edwin Romijn and Prof. John R. Yates III, who introduce the different analytical strategies developed and successfully utilized to study organelle proteomes, and detail the use of multidimensional liquid chromatography coupled to tandem mass spectrometry for peptide sample analysis. This book is further composed of two main sections. First, detailed protocols are provided to perform the purification of the various organelles present in eukaryotic cells, as well as to prepare certain sub-fractions of organelles (chapters 2-22). In all cases, the samples are aimed to be analyzed by a mass spectrometry technique. While an exhaustive list of chapters covering all the proteomic analyses of organelles and organelle fractions was not conceivable, we nevertheless wanted to provide analysis examples reflecting the trend toward more specific purifications of organelle sub-fractions, which will allow reaching the more comprehensive and accurate characterization of the organelle. Most of the chapters cover the whole analytical procedure of organelle characterization, from its purification starting with whole cells up to protein identification using mass spectrometry. In some cases, the chapter may provide a detailed de@@@@@@ion of the purification process wherein less classical techniques appear which are implemented by a minority of laboratories (e.g. free flow electrophoresis). Second, however optimized the organelle purification protocol and skilled the operator the sample of interest will never consist of the pure targeted organelle. Therefore, among the proteins identified, one has to separate the true from the intruders. The actual sub-cellular localization of some individual proteins newly attributed to the studied organelle can be evaluated by orthogonal assays, such as microscopy, by expressing the GFP-tagged version of the protein candidates. Yet this approach is labor-intensive and is usually restricted to a few selected proteins. We devoted the second section of this book to methods enabling a global estimate of the reliability of the protein list assigned to an organelle. An average ratio of proteins wrongly attributed to the organelle of interest is provided by assessing sample purity (chapter 23). In order to determine whether every identified protein is an actual component of the purified organelle quantitative mass spectrometry methods can be employed (chapters 24-26). In chapter 26, Dr. Wei Yan et al. more specifically demonstrate the utility of quantitative approaches to scrutinize protein shuttling between organelles. The examples presented of quantitative mass spectrometry analysis of organelle fractions use a few commercially available isotope-tagged reagents, but many other chemicals, either commercial or prepared in-house, can be utilized. A larger variety of the existing polypeptide labeling strategies can be found in another volume of this series entitled Quantitative Proteomics, edited by Dr. Salvatore Sechi. Finally, the last chapter of this book, by Dr. Wallace F. Marshall, addresses the use of tran@@@@@@omic data to identify genes potentially encoding organelle proteomes.
Human genome sequencing has identified about 25000 genes, most being of unknown function. Localization of the final gene products, i.e. the proteins, in a specific organelle is a key to decipher the proteins roles within the cell. Over the past twenty years, proteomic analyses have progressively proved to be an invaluable tool to obtain high-throughput protein identification from low-abundance, complex biological samples. These analyses boomed thanks to dramatic technological progresses in mass spectrometry instrumentation, optimization of its coupling to capillary liquid chromatography and the development of software enabling processing of the vast amount of generated data. In the context of organelle study, such analyses have allowed greater depth in the characterization of the proteins constitutive of, or transiently present in, these large functional modules.
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