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Jennifer Elizabeth Grant Hong Li Editors Analysis of Post-Translational Modi Cations and Proteolysis in Neuroscience N EUROMETHODS Neuromethods 114 Jennifer Elizabeth Grant Hong Li Editors Analysis of Post-Translational Modi cations and Proteolysis in Neuroscience N EUROMETHODS Series Editor Wolfgang Walz University of Saskatchewan Saskatoon, SK, Canada For further volumes: http://www.springer.com/series/7657 Analysis of Post-Translational Modifications and Proteolysis in Neuroscience Edited by Jennifer Elizabeth Grant Department of Biology University of Wisconsin-Stout Menomonie, Wisconsin, USA Hong Li Department of Microbiology, Biochemistry and Molecular Genetics Rutgers University—New Jersey Medical School Cancer Center Newark Newark, NJ, USA Editors Jennifer Elizabeth Grant Hong Li Department of Biology Department of Microbiology, Biochemistry University of Wisconsin-Stout and Molecular Genetics Menomonie, Wisconsin, USA Rutgers University—New Jersey Medical School Cancer Center Newark Newark, NJ, USA ISSN 0893-2336 ISSN 1940-6045 (electronic) Neuromethods ISBN 978-1-4939-3470-6 ISBN 978-1-4939-3472-0 (eBook) DOI 10.1007/978-1-4939-3472-0 Library of Congress Control Number: 2016946421 © Springer Science+Business Media New York 2016 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC New York Preface to the Series Experimental life sciences have two basic foundations: concepts and tools. The Neuro- methods series focuses on the tools and techniques unique to the investigation of the nervous system and excitable cells. It will not, however, shortchange the concept side of things as care has been taken to integrate these tools within the context of the concepts and questions under investigation. In this way, the series is unique in that it not only collects protocols but also includes theoretical background information and critiques which led to the methods and their development. Thus it gives the reader a better understanding of the origin of the techniques and their potential future development. The Neuromethods publishing program strikes a balance between recent and exciting developments like those concerning new animal models of disease, imaging, in vivo methods, and more established techniques, including, for example, immunocytochemistry and electrophysiological technologies. New trainees in neurosciences still need a sound footing in these older methods in order to apply a critical approach to their results. Under the guidance of its founders, Alan Boulton and Glen Baker, the Neuromethods series has been a success since its first volume published through Humana Press in 1985. The series continues to flourish through many changes over the years. It is now published under the umbrella of Springer Protocols. While methods involving brain research have changed a lot since the series started, the publishing environment and technology have changed even more radically. Neuromethods has the distinct layout and style of the Springer Protocols program, designed specifically for readability and ease of reference in a laboratory setting. The careful application of methods is potentially the most important step in the process of scientific inquiry. In the past, new methodologies led the way in developing new dis- ciplines in the biological and medical sciences. For example, Physiology emerged out of Anatomy in the nineteenth century by harnessing new methods based on the newly discov- ered phenomenon of electricity. Nowadays, the relationships between disciplines and meth- ods are more complex. Methods are now widely shared between disciplines and research areas. New developments in electronic publishing make it possible for scientists that encounter new methods to quickly find sources of information electronically. The design of individual volumes and chapters in this series takes this new access technology into account. Springer Protocols makes it possible to download single protocols separately. In addition, Springer makes its print-on-demand technology available globally. A print copy can therefore be acquired quickly and for a competitive price anywhere in the world. Saskatoon, Canada Wolfgang Walz v Preface Analysis of Post-translational Modifications and Proteolysis in Neuroscience is designed to provide detailed experimental procedures for proteomics studies of post-translational mod- ifications (PTMs) to answer basic and translational research questions in neuroscience. Our main focus is on recent studies that apply state-of-the-art mass spectrometry techniques for either qualitative identifications of PTMs or the determination of their dynamics in neuronal cells and tissues. That said, we have also included additional cutting-edge approaches that may benefit future neuroscience studies. In many cases, the chapters presented here include novel and sophisticated examples of targeted proteomics, where much creativity is employed in the sample work-up. Given that space is held at a premium in journal articles, some of the technical intricacies are not always described precisely within the methods and protocols sections, complicating the efforts of others to either reproduce the results or apply similar methods to study PTMs in neuronal cells and tissues. We hope this collection of articles will fill this gap and provide sufficient details that will help readers to successfully conduct similar experiments in their labs. Applications utilizing proteomics to answer neuroscience questions have made tremen- dous progress. These approaches are becoming more effective due to significant improve- ment in the sensitivity of mass spectrometers and the sophistication of affiliated bioinformatics software. The recent availability of the Orbitrap and Triple-TOF mass spectrometers has revolutionized protein detection sensitivities to low attomole levels, while the development of electron transfer dissociation (ETD) has allowed preservation of PTMs that often degraded during MS/MS fragmentation. On the digital end, development of MaxQuant and other software packages for rapid analysis of either unlabeled or stable isotope-labeled phosphopeptides has greatly enabled our ability to understand protein- signaling networks in cells and tissues. Furthermore, state-of-the-art software such as Skyline has made targeted quantification of selected proteins in a large number of biological samples much more feasible. Yet, despite this progress, neuroproteomicists must still confront multiple layers of complexity associated with neurological processes and structures. In both the central and peripheral nervous systems, many cells types form intricate networks, highly intertwined, to accomplish the complex biological tasks such as the ones associated with memory and thought formation, judgment, and neuromuscular activity. In this complex milieu it is very difficult to dissect cell-specific functions, where contributions of the neighboring cells often obfuscate analysis. An additional complication is that proteomes are more complex compared to genomes, where a diverse array of dynamic processes regulate PTMs, including proteolysis. Such post-translational activities are crucial for maintaining protein function, enzymatic activities, protein subcellular localization, protein-protein interactions, and even protein turnover. On a systems level, abnormally modified and processed proteins have been implicated to be accumulated in brains of patients suffering from many neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and prion diseases among others. In addition, proteins with rare PTMs, including citrullination, have been shown to be associated with increased autoimmunity in multiple sclerosis and other degenerative neuronal diseases. Therefore, it is essential to be able to accurately identify vii viii Preface PTMs and regulated proteolysis in neuronal proteins and quantify their dynamic changes during normal signaling events, and during disease progression. Each individual chapter in Analysis of Post-translational Modifications and Proteolysis in Neuroscience is designed to provide detailed information regarding the analysis of protein processing and PTMs, as well as notes that draw attention to key steps that require extra care for the successful execution of the experiment. The methods described here are contributed by experts in neuroproteomics, most of whom published their original research on proteins derived from neuronal cells and tissues in peer-reviewed journals.
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