Synopses & Reviews
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 description 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 a and skilled the operator a 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 actualsub-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 transcriptomic 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 roleswithin 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.
Synopsis
Proteomic analyses have proven to be an invaluable tool in obtaining high-throughput protein identification from low-abundance, complex biological samples. This is the first book that examines organelle proteomics in depth. Detailed protocols provide step-by-step instructions to successfully study organelle proteomes by performing the purification of the various organelles present in eukaryotic cells, as well as by preparing certain sub-fractions of organelles. A series of chapters cover the whole analytical procedure of organelle characterization, from its purification starting with whole cells up to protein identification using mass spectrometry. Also, devoted to methods enabling a global estimate of the reliability of the protein list assigned to an organelle, the book allows scientists to gain a vital and important understanding of organelle study.
Synopsis
Proteomic analyses have proven to be an invaluable tool in obtaining high-throughput protein identification from low-abundance, complex biological samples. In Organelle Proteomics, detailed protocols provide step-by-step instructions to successfully study organelle proteomes by performing the purification of the various organelles present in eukaryotic cells, as well as by preparing certain sub-fractions of organelles. A series of chapters cover the whole analytical procedure of organelle characterization, from its purification starting with whole cells up to protein identification using mass spectrometry. Devoted to methods enabling a global estimate of the reliability of the protein list assigned to an organelle, Organelle Proteomics allows scientists to gain a vital and important understanding of organelle study.
Synopsis
This is the first book to examine organelle proteomics in depth. It begins by introducing the different analytical strategies developed and successfully utilized to study organelle proteomes, and detailing the use of multidimensional liquid chromatography coupled to tandem mass spectrometry for peptide sample analysis. Detailed protocols are provided and a section is devoted to methods enabling a global estimate of the reliability of the protein list assigned to an organelle.
Table of Contents
1. - Analysis of organelles by on-line two-dimensional liquid chromatography - tandem mass spectrometry (2DLC-MS/MS) Edwin P. Romijn and John R. Yates III. Analysis of organelles, vesicles and protein machineries 2 - Purification and proteomic analysis of chloroplasts and their sub-organellar compartments Daniel Salvi, Norbert Rolland, Jacques Joyard and Myriam Ferro 3 - Proteomic analysis of the stacked Golgi complex Mark S. Lowenthal, Kathryn E. Howell, and Christine C. Wu 4 - Purification of Saccharomyces cerevisiae mitochondria by zone-electrophoresis in a free flow device (ZE-FFE) Hans Zischka, Norbert Kinkl, Ralf J. Braun and Marius Ueffing 5 - Preparation of Respiratory Chain Complexes from Saccharomyces cerevisiae Wild Type and Mutant Mitochondria: Activity Measurement and Subunit Composition Analysis Claire Lemaire and Geneviève Dujardin 6 - Mitochondrial proteomics: Analysis of a whole mitochondrial extract with two-dimensional electrophoresis Thierry Rabilloud. 7 - Purification and proteomic analysis of the mouse liver mitochondrial inner membrane Sandrine Da Cruz and Jean-Claude Martinou 8 - Subcellular Fractionation and Proteomics of Nuclear Envelopes Laurence Florens, Nadia Korfali and Eric C. Schirmer. 9 - Purification and proteomic analysis of the nuclear insoluble protein fraction Tsuneyoshi Horigome, Kazuhiro Furukawa and Kohei Ishii 10 - Preparation Methods of Human Metaphase Chromosomes for Their Proteome Analysis. Kiichi Fukui and Hideaki Takata 11 - Purification and proteomic analysis of plant plasma membranes Erik Alexandersson, Niklas Gustavsson, Katja Bernfur, Adine Karlsson, Per Kjellbom and Christer Larsson 12 - Protocol to enrich and analyze plasma membrane proteins from frozen tissue Jacek R. Wisniewski 13 - Proteomic characterization of membrane protein topology Adele R. Blackler and Christine C. Wu 14 - Free Flow Isoelectric Focussing (FF-IEF): A method for the separation of both hydrophilic and hydrophobic proteins of rat liver peroxisomes Markus Islinger and Gerhard Weber 15 - Use of gas-phase-fractionation to increase protein identifications: application to the peroxisome Jacob Kennedy and Eugene C. Yi 16 - Purification and proteomic analysis of lysosomal integral membrane proteins Huiwen Zhang, Xiaolian Fan, Rick Bagshaw, Don J. Mahuran, John W. Callahan. 17 - Affinity purification of soluble lysosomal proteins for mass spectrometric identification Sylvie Kieffer-Jaquinod, Agnès Chapel, Jérôme Garin and Agnès Journet 18 - Purification and proteomic analysis of synaptic vesicles Holly D. Cox and Charles M. Thompson. 19 - Purification and proteomic analysis of pancreatic zymogen granule membranes Xuequn Chen and Philip C. Andrews 20 - Isolation and proteomic analysis of Chlamydomonas centrioles Lani C. Keller and Wallace F. Marshall. 21 - Purification and proteomic analysis of 20S proteasomes from human cells Marie-Pierre Bousquet-Dubouch, Sandrine Uttenweiler-Joseph, Manuelle Ducoux-Petit, Mariette Matondo, Bernard Monsarrat, and Odile Burlet-Schiltz. 22 - Characterization of E. coli ribosomal particles: combined analysis of whole proteins by mass spectrometry and of proteolytic digests by liquid chromatography - tandem mass spectrometry. Isabelle Iost, Julie Charollais, Joëlle Vinh and Delphine Pflieger Proteins identified in the organelle sample: true components or contaminants? 23 - Assessment of organelle purity using antibodies and specific assays: the example of the chloroplast envelope Daniel Salvi, Norbert Rolland, Jacques Joyard and Myriam Ferro 24 - Identifying bona fide components of an organelle by isotope coded labelling of subcellular fractions: an example in peroxisomes Marcello Marelli, Alexey I. Nesvizhskii and John D. Aitchison 25 - Determination of Genuine Residents of Plant Endomembrane Organelles using Isotope Tagging and Multivariate Statistics Kathryn S. Lilley and Tom P.J. Dunkley 26. - Quantitative Proteomic Analysis to Profile Dynamic Changes in the Spatial Distribution of Cellular Proteins Wei Yan, Daehee Hwang, Ruedi Aebersold 27 - Use of transcriptomic data to support organelle proteomic analysis Wallace F. Marshall