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NEUROPROTEOMICS
FRONTIERS IN NEUROSCIENCE
Series Editors
Sidney A. Simon, Ph.D.
Miguel A.L. Nicolelis, M.D., Ph.D.

Published Titles
Apoptosis in Neurobiology
Yusuf A. Hannun, M.D., Professor of Biomedical Research and Chairman, Department
of Biochemistry and Molecular Biology, Medical University of South Carolina,
Charleston, South Carolina
Rose-Mary Boustany, M.D., tenured Associate Professor of Pediatrics and Neurobiology,
Duke University Medical Center, Durham, North Carolina

Neural Prostheses for Restoration of Sensory and Motor Function

John K. Chapin, Ph.D., Professor of Physiology and Pharmacology, State University
of New York Health Science Center, Brooklyn, New York
Karen A. Moxon, Ph.D., Assistant Professor, School of Biomedical Engineering, Science,
and Health Systems, Drexel University, Philadelphia, Pennsylvania

Computational Neuroscience: Realistic Modeling for Experimentalists

Eric DeSchutter, M.D., Ph.D., Professor, Department of Medicine, University of Antwerp,
Antwerp, Belgium

Methods in Pain Research

Lawrence Kruger, Ph.D., Professor of Neurobiology (Emeritus), UCLA School of Medicine
and Brain Research Institute, Los Angeles, California

Motor Neurobiology of the Spinal Cord

Timothy C. Cope, Ph.D., Professor of Physiology, Wright State University, Dayton, Ohio

Nicotinic Receptors in the Nervous System

Edward D. Levin, Ph.D., Associate Professor, Department of Psychiatry and Pharmacology
and Molecular Cancer Biology and Department of Psychiatry and Behavioral Sciences,
Duke University School of Medicine, Durham, North Carolina

Methods in Genomic Neuroscience

Helmin R. Chin, Ph.D., Genetics Research Branch, NIMH, NIH, Bethesda, Maryland
Steven O. Moldin, Ph.D., University of Southern California, Washington, D.C.

Methods in Chemosensory Research

Sidney A. Simon, Ph.D., Professor of Neurobiology, Biomedical Engineering,
and Anesthesiology, Duke University, Durham, North Carolina
Miguel A.L. Nicolelis, M.D., Ph.D., Professor of Neurobiology and Biomedical Engineering,
Duke University, Durham, North Carolina

The Somatosensory System: Deciphering the Brain’s Own Body Image

Randall J. Nelson, Ph.D., Professor of Anatomy and Neurobiology,
University of Tennessee Health Sciences Center, Memphis, Tennessee
The Superior Colliculus: New Approaches for Studying Sensorimotor Integration
William C. Hall, Ph.D., Department of Neuroscience, Duke University,
Durham, North Carolina
Adonis Moschovakis, Ph.D., Department of Basic Sciences, University of Crete,
Heraklion, Greece

New Concepts in Cerebral Ischemia

Rick C. S. Lin, Ph.D., Professor of Anatomy, University of Mississippi Medical Center,
Jackson, Mississippi

DNA Arrays: Technologies and Experimental Strategies

Elena Grigorenko, Ph.D., Technology Development Group, Millennium Pharmaceuticals,
Cambridge, Massachusetts

Methods for Alcohol-Related Neuroscience Research

Yuan Liu, Ph.D., National Institute of Neurological Disorders and Stroke,
National Institutes of Health, Bethesda, Maryland
David M. Lovinger, Ph.D., Laboratory of Integrative Neuroscience, NIAAA,
Nashville, Tennessee

Primate Audition: Behavior and Neurobiology

Asif A. Ghazanfar, Ph.D., Princeton University, Princeton, New Jersey

Methods in Drug Abuse Research: Cellular and Circuit Level Analyses

Dr. Barry D. Waterhouse, Ph.D., MCP-Hahnemann University, Philadelphia, Pennsylvania

Functional and Neural Mechanisms of Interval Timing

Warren H. Meck, Ph.D., Professor of Psychology, Duke University, Durham, North Carolina

Biomedical Imaging in Experimental Neuroscience

Nick Van Bruggen, Ph.D., Department of Neuroscience Genentech, Inc.
Timothy P.L. Roberts, Ph.D., Associate Professor, University of Toronto, Canada

The Primate Visual System

John H. Kaas, Department of Psychology, Vanderbilt University
Christine Collins, Department of Psychology, Vanderbilt University, Nashville, Tennessee

Neurosteroid Effects in the Central Nervous System

Sheryl S. Smith, Ph.D., Department of Physiology, SUNY Health Science Center,
Brooklyn, New York

Modern Neurosurgery: Clinical Translation of Neuroscience Advances

Dennis A. Turner, Department of Surgery, Division of Neurosurgery,
Duke University Medical Center, Durham, North Carolina

Sleep: Circuits and Functions

Pierre-Hervé Luoou, Université Claude Bernard Lyon, France

Methods in Insect Sensory Neuroscience

Thomas A. Christensen, Arizona Research Laboratories, Division of Neurobiology,
University of Arizona, Tuscon, Arizona

Motor Cortex in Voluntary Movements

Alexa Riehle, INCM-CNRS, Marseille, France
Eilon Vaadia, The Hebrew University, Jerusalem, Israel
Neural Plasticity in Adult Somatic Sensory-Motor Systems
Ford F. Ebner, Vanderbilt University, Nashville, Tennessee

Advances in Vagal Afferent Neurobiology

Bradley J. Undem, Johns Hopkins Asthma Center, Baltimore, Maryland
Daniel Weinreich, University of Maryland, Baltimore, Maryland

The Dynamic Synapse: Molecular Methods in Ionotropic Receptor Biology

Josef T. Kittler, University College, London, England
Stephen J. Moss, University College, London, England

Animal Models of Cognitive Impairment

Edward D. Levin, Duke University Medical Center, Durham, North Carolina
Jerry J. Buccafusco, Medical College of Georgia, Augusta, Georgia

The Role of the Nucleus of the Solitary Tract in Gustatory Processing

Robert M. Bradley, University of Michigan, Ann Arbor, Michigan

Brain Aging: Models, Methods, and Mechanisms

David R. Riddle, Wake Forest University, Winston-Salem, North Carolina

Neural Plasticity and Memory: From Genes to Brain Imaging

Frederico Bermudez-Rattoni, National University of Mexico, Mexico City, Mexico

Serotonin Receptors in Neurobiology

Amitabha Chattopadhyay, Center for Cellular and Molecular Biology, Hyderabad, India

Methods for Neural Ensemble Recordings, Second Edition

Miguel A.L. Nicolelis, M.D., Ph.D., Professor of Neurobiology and Biomedical Engineering,
Duke University Medical Center, Durham, North Carolina

Biology of the NMDA Receptor

Antonius M. VanDongen, Duke University Medical Center, Durham, North Carolina

Methods of Behavioral Analysis in Neuroscience

Jerry J. Buccafusco, Ph.D., Alzheimer’s Research Center, Professor of Pharmacology
and Toxicology, Professor of Psychiatry and Health Behavior,
Medical College of Georgia, Augusta, Georgia

In Vivo Optical Imaging of Brain Function, Second Edition

Ron Frostig, Ph.D., Professor, Department of Neurobiology,
University of California, Irvine, California

Fat Detection: Taste, Texture, and Post Ingestive Effects

Jean-Pierre Montmayeur, Ph.D., Centre National de la Recherche Scientifique, Dijon, France
Johannes le Coutre, Ph.D., Nestlé Research Center, Lausanne, Switzerland

The Neurobiology of Olfaction

Anna Menini, Ph.D., Neurobiology Sector International School for Advanced
Studies,(S.I.S.S.A.), Trieste, Italy

Neuroproteomics
Oscar Alzate, Ph.D., Department of Cell and Developmental Biology, University of North
Carolina, Chapel Hill, North Carolina
NEUROPROTEOMICS
Edited by
Oscar Alzate
University of North Carolina

Boca Raton London New York

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Library of Congress Cataloging‑in‑Publication Data

Neuroproteomics / editor, Oscar Alzate.

p. ; cm. -- (Frontiers in neuroscience)
Includes bibliographical references and index.
ISBN 978-1-4200-7625-7 (hardcover : alk. paper)
1. Proteomics. 2. Nervous system--Diseases--Genetic aspects. I. Alzate, Oscar. II.
Series: Frontiers in neuroscience (Boca Raton, Fla.)
[DNLM: 1. Proteomics--methods. 2. Nervous System Diseases--metabolism.
3. Nervous System Physiological Phenomena--genetics. 4. Proteome--isolation &
purification. QU 58.5 N494 2010]

QP551.N485 2010
572’.6--dc22 2009031460

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and the CRC Press Web site at

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Contents
Series Preface.............................................................................................................ix
Foreword....................................................................................................................xi
Preface.................................................................................................................... xiii
Editor........................................................................................................................ xv
Contributors............................................................................................................xvii

Chapter 1 Neuroproteomics................................................................................... 1
Oscar Alzate

Chapter 2 Banking Tissue for Neurodegenerative Research............................... 17

John F. Ervin

Chapter 3 Multidimensional Techniques in Protein Separations for

Neuroproteomics.................................................................................25
Carol Haney Ball and Petra Levine Roulhac

Chapter 4 2-D Fluorescence Difference Gel Electrophoresis (DIGE) in

Neuroproteomics................................................................................. 51
Roberto Diez, Michael Herbstreith, Cristina Osorio, and
Oscar Alzate

Chapter 5 Mass Spectrometry for Proteomics..................................................... 71

Carol E. Parker, Maria R. Warren, and Viorel Mocanu

Chapter 6 Mass Spectrometry for Post-Translational Modifications................... 93

Carol E. Parker, Viorel Mocanu, Mihaela Mocanu,
Nedyalka Dicheva, and Maria R. Warren

Chapter 7 MALDI Imaging and Profiling Mass Spectrometry in

Neuroproteomics............................................................................... 115
Malin Andersson, Per Andren, and Richard M. Caprioli

Chapter 8 Protein Interaction Networks............................................................ 135

Alexei Vazquez

vii
viii Contents

Chapter 9 Knowledge-Based Analysis of Protein Interaction Networks in

Neurodegenerative Diseases.............................................................. 147
Minoru Kanehisa, Vachiranee Limviphuvadh, and Mao Tanabe

Chapter 10 Redox Proteomics of Oxidatively Modified Brain Proteins in

Mild Cognitive Impairment.............................................................. 163
Tanea T. Reed, Rukhsana Sultana, and D. Allan Butterfield

Chapter 11 Neuroproteomics in the Neocortex of Mammals: Molecular

Fingerprints of Cortical Plasticity..................................................... 197
Lieselotte Cnops, Tjing-Tjing Hu, Gert Van den Bergh, and
Lutgarde Arckens

Chapter 12 A Neuroproteomic Approach to Understanding Visual Cortical

Development...................................................................................... 215
Leonard E. White

Chapter 13 Behaviorally Regulated mRNA and Protein Expression in the

Songbird Brain.................................................................................. 239
Miriam V. Rivas and Erich D. Jarvis

Chapter 14 Proteomics of Experience–Dependent Plasticity in the Songbird

Auditory Forebrain: Quantitative Proteomic Analyses of
Experience-Regulated Proteins......................................................... 263
Raphael Pinaud, Oscar Alzate, and Liisa A. Tremere

Chapter 15 Applications of Proteomics to Nerve Regeneration Research.......... 289

Mark W. Massing, Grant A. Robinson, Christine E. Marx,
Oscar Alzate, and Roger D. Madison

Index....................................................................................................................... 315
Series Preface
Our goal in creating the Frontiers in Neuroscience Series is to present the insights
of experts on emerging fields and theoretical concepts that are, or will be, in
the vanguard of neuroscience. Books in the series cover genetics, ion channels,
apoptosis, electrodes, neural ensemble recordings in behaving animals, and
even robotics. The series also covers new and exciting multidisciplinary areas of
brain research, such as computational neuroscience and neuroengineering, and
describes breakthroughs in classical fields like behavioral neuroscience. We hope
every neuroscientist will use these books in order to get acquainted with new ideas
and frontiers in brain research. These books can be given to graduate students
and postdoctoral fellows when they are looking for guidance to start a new line
of research.
Each book is edited by an expert and consists of chapters written by the leaders
in a particular field. Books are richly illustrated and contain comprehensive bibliog-
raphies. Chapters provide substantial background material relevant to the particu-
lar subject. We hope that as the volumes become available, the effort put in by us,
the publisher, the book editors, and individual authors will contribute to the further
development of brain research. The extent to which we achieve this goal will be
determined by the utility of these books.

Sidney A. Simon, Ph.D.

Miguel A. L. Nicolelis, M.D., Ph.D.
Series Editors

ix
Foreword
My first exposure to experimental biology involved a college job, when I worked in
the lab of Dr. Norman Anderson in Oak Ridge, Tennessee. He had recently launched
his “Molecular Anatomy” (MAN) project, arguably the first step toward contempo-
rary proteomics. I enjoyed my summer, working as a junior lackey in an ultrastruc-
tural core facility that supported a component of the project involving centrifugal
fractionation of tissue homogenates. (Curiously, our group’s main task was to help
develop better vaccines for influenza.) I note, 40 years later, that the first citation
in Chapter 1 of Neuroproteomics is to Norman Anderson. I suppose this anecdote
points both to the vision of Dr. Anderson and to the impact that a summer job can
have on a college student.
My current research focuses on the arrangement of proteins in excitatory syn-
apses of the rodent forebrain. Over my career I have been repeatedly astonished by
the complexity of individual synapses and by the precision with which their numer-
ous components are organized. This embodies one of the joys of neuroscience, but
at the same time perhaps its greatest problem: Given the overwhelming complexities
that suffuse every level of the nervous system, how can we hope to gain a real under-
standing of the brain? This goal is one of the few truly difficult problems of natural
science. While acknowledging the magnitude of the task, perhaps we should instead
marvel that so much has already been discovered.
Neuroscience has advanced thanks to a great deal of hard work, along with
clear thinking and a bit of luck; but equally important has been the develop-
ment of a large and growing technical toolbox. Indeed, it could be argued that
the most important advances in neuroscience have rested on the introduction of
powerful new tools. Dating back at least to the introduction of the oscilloscope,
tools from the physical sciences have had a disproportionate impact on neuro-
science. It is in this context that Professor Alzate’s book is especially welcome.
Neuroproteomics addresses some of the simplest and most straightforward ques-
tions about the brain, but these questions remain unanswered. Proteomic tools
can offer valuable insights into a wide variety of crucial issues ranging from
development to the synaptic plasticity that underlies learning and memory to the
basis of neuropsychiatric disease. Recent work in neuroproteomics has also led to
entirely novel “systems biology” ways to think about the nervous system (see, for
example, http://www.genes2cognition.org).
By assembling a full spectrum of neuroproteomic techniques and applications within
a single volume, Dr. Alzate has simplified the practical tasks facing a neurobiologist
who needs an introduction to proteomic methods. Moreover, this volume will enable
the neuroscience community as a whole to recognize neuroproteomics as an important
new tool for the entire field.

Richard Weinberg, PhD

University of North Carolina at Chapel Hill

xi
Preface
Neuroproteomics is gaining momentum. The use of proteomics to elucidate brain
phenomena is a natural result of the human’s interest in his “thinking organ” and the
ever-increasing number of modern techniques for the systematic analysis of proteins
and protein interaction networks. Moreover, it is in the brain that the protein interac-
tion networks are at the center stage of the scientific inquiry, as their physico-chem-
ical interactions result in phenomena such as learning, memory, logic interpretation
and production of information, visualization, coordination of motion, body orienta-
tion, and many others. Neuroproteomics takes advantage of the advent of multiple
techniques for isolation, identification, and characterization of large numbers of pro-
teins, and the subsequent analysis of their localization, molecular interactions, and
participation in regulatory and metabolic pathways.
Pursuing this broad aim, neuroproteomics uses multiple approaches such as mass
spectrometry, electrophoresis, chromatography, surface plasmon resonance, protein
arrays, immunoblotting, computational proteomics, and molecular imaging to help
elucidate the roles of proteins in the different parts of the nervous system. And this
is only the beginning. Many challenges wait ahead for neuroproteomics, including
identification of these proteins and their corresponding localization, characteriza-
tion of protein conformations and post-translational modifications, molecular inter-
actions involving proteins and other molecules, identification of the regulatory and
metabolic networks in which these proteins are involved, and ultimately the identifi-
cation of potential methods to regulate the structures and functions of these protein
interaction networks. Finally, neuroproteomics has to explain how this vast amount
of protein data changes with time and with external conditions, giving origin to brain
functions. This ultimate goal will permit great advances in the understanding of the
brain, its functions, its structure, and how deviations from normal states lead to neu-
rological and mental diseases.
This book is the result of several years of work using proteomics to understand
biological processes in the nervous system. Proteomics studies large ensembles of
proteins and requires many techniques aimed at understanding how proteins interact
with each other and with other molecules within the ensemble, and how proteins
function in the context of their respective environments and under specific condi-
tions. Investigators using neuroproteomics need to start with very intricate sample
preparation, and proceed to mass spectrometry, to HPLC, to 2D-DIGE, to protein
arrays, or to protein interaction networks. They need to correlate this informa-
tion with action potentials, electrical signals, and synaptic activity. Each of these
approaches has its own strengths and limitations, and all are needed to achieve a
reliable interpretation of neurobiological processes.
This volume provides an overview of what neuroproteomics is, and gives a
few examples of what neuroproteomics does. The general idea was to present the
principles, the approaches, the difficulties of each technique, and the challenges of
the field. Some of the authors have laboratories dedicated to the investigation of

xiii
xiv Preface

neurobiological phenomena, and have integrated proteomics approaches to elucidate

relevant biological functions of the nervous system. Other authors are experts in
specific approaches that can be used for the study of neuroproteomics.
I would like to acknowledge the help that I received from all the authors. It has
been a difficult task to put together all of the information required—in many cases,
data were collected specifically for inclusion in this book. I want to extend special
thanks to Sid Simon and Miguel Nicolelis for inviting me to undertake this project—
it has been an enriching experience. I also want to thank Cristina Osorio for her
help in contacting the authors, collecting all of the manuscripts, and for her personal
support and assistance with this project, starting from the very beginning. Finally, I
want to express my appreciation for the dedication and professionalism of the staff
of Taylor & Francis, particularly Barbara Norwitz, Patricia Roberson, and Prudence
Board. Thanks to each one of you.

Oscar Alzate, Ph.D.

Editor
Dr. Oscar Alzate currently holds the position of associate professor in the Department
of Cell and Developmental Biology at the University of North Carolina (Chapel Hill,
North Carolina). He is also the director of the UNC Systems Proteomics Center. His
interests include the application of neuroproteomics to the elucidation of molecular
pathways and protein interaction networks using animal models for neurodegenera-
tive diseases, particularly Alzheimer’s disease. Dr. Alzate, a molecular biophysi-
cist, has developed proteomics laboratories for the Davis Heart and Lung Research
Institute at the Ohio State University, the Neuroproteomics Laboratory at Duke
University, and the Proteomics Laboratory at the Pontifical Bolivariana University
in his native Colombia. His passion is playing with proteins, trying to develop better
ways to isolate, identify, and characterize proteins using combinations of all avail-
able biophysical techniques.
Dr. Alzate’s search for functional protein interaction networks in cell cultures,
primary cells, and mouse and human tissue in order to develop models of neu-
rological diseases utilizes differential-display proteomics, mass spectrometry,
iTRAQ, protein arrays, MALDI-based tissue imaging, and computational pro-
teomics. In addition to his own research program, he is involved in more than
20 collaborative projects that include the neurobiology of synapses; neuropro-
teomics of the auditory and visual systems; and Alzheimer’s, Parkinson’s, and
Huntington’s diseases, as well as epilepsy and amyotrophic lateral sclerosis.

xv
Contributors
Oscar Alzate, Ph.D. Richard M. Caprioli, Ph.D.
Department of Cell and Developmental Department of Biochemistry
Biology Mass Spectrometry Research Center
Program in Molecular Biology and Vanderbilt University Medical Center
Biotechnology Nashville, Tennessee
University of North Carolina at
Chapel Hill
Lieselotte Cnops, Ph.D.
Chapel Hill, North Carolina
Laboratory of Neuroplasticity and
Malin Andersson, Ph.D. Neuroproteomics
Department of Pharmaceutical Department of Biology
Biosciences Katholieke Universiteit Leuven
Medical Mass Spectrometry Leuven, Belgium
Uppsala University
Uppsala, Sweden Nedyalka Dicheva, M.S.
Per Andren, Ph.D. UNC Duke Proteomics Center
Department of Pharmaceutical Program in Molecular Biology and
Biosciences Biotechnology
Medical Mass Spectrometry University of North Carolina at
Uppsala University Chapel Hill
Uppsala, Sweden Chapel Hill, North Carolina

Lutgarde Arckens, Ph.D.

Roberto Diez, M.Sc.
Laboratory of Neuroplasticity and
GE Healthcare Life Sciences
Neuroproteomics
Department of Biology Piscataway, New Jersey
Katholieke Universiteit Leuven
Leuven, Belgium John F. Ervin
Kathleen Price Bryan Brain Bank
Carol Haney Ball, Ph.D. Duke University Medical Center
Agilent Technologies Division of Neurology
Cary, North Carolina
Durham, North Carolina
D. Allan Butterfield, Ph.D.
Department of Chemistry Michael Herbstreith, B.Sc.
Center of Membrane Sciences and Department of Medicine
Sanders-Brown Center on Aging Division of Neurology
University of Kentucky Duke University Medical Center
Lexington, Kentucky Durham, North Carolina

xvii
xviii Contributors

Tjing-Tjing Hu, Ph.D. Mark W. Massing, Ph.D.

Laboratory of Neuroplasticity and Research Service of the Veterans
Neuroproteomics Affairs Medical Center
Department of Biology Durham, North Carolina
Katholieke Universiteit Leuven
Leuven, Belgium Mihaela Mocanu, B.S.
UNC Duke Proteomics Center
Erich D. Jarvis, Ph.D. Program in Molecular Biology and
Department of Neurobiology Biotechnology
Duke University Medical Center University of North Carolina at
Durham, North Carolina Chapel Hill
Chapel Hill, North Carolina
Minoru Kanehisa, Ph.D.
Bioinformatics Center Viorel Mocanu, Ph.D.
Institute for Chemical Research UNC Duke Proteomics Center
Kyoto University Program in Molecular Biology and
Kyoto, Japan, and Biotechnology
University of North Carolina at
Human Genome Center
Chapel Hill
Institute of Medical Science
Chapel Hill, North Carolina
The University of Tokyo
Tokyo, Japan
Cristina Osorio, B.Sc.
Program on Molecular Biology and
Vachiranee Limviphuvadh, Ph.D.
Biotechnology
Bioinformatics Center
School of Medicine
Institute for Chemical Research
University of North Carolina at
Kyoto University
Chapel Hill
Kyoto, Japan Chapel Hill, North Carolina

Roger D. Madison, Ph.D. Carol E. Parker, Ph.D.

Research Service UNC Duke Proteomics Center
Veterans Affairs Medical Center Program in Molecular Biology and
Department of Surgery Biotechnology
Department of Neurobiology Department of Biochemistry and
Duke University Medical Center Biophysics
Durham, North Carolina University of North Carolina at
Chapel Hill
Christine E. Marx, Ph.D. Chapel Hill, North Carolina
Research Service
Veterans Affairs Medical Center Raphael Pinaud, Ph.D.
Department of Surgery Department of Brain and Cognitive
Department of Neurobiology Sciences
Duke University Medical Center University of Rochester
Durham, North Carolina Rochester, New York
Contributors xix

Tanea T. Reed, Ph.D. Liisa A. Tremere, Ph.D.

Department of Chemistry Department of Brain and Cognitive
Eastern Kentucky University Sciences
Richmond, Kentucky University of Rochester
Rochester, New York
Miriam V. Rivas, Ph.D.
Department of Neurobiology Gert Van den Bergh, Ph.D.
Duke University Medical Center Laboratory of Neuroplasticity and
Durham, North Carolina Neuroproteomics
Department of Biology
Grant A. Robinson, Ph.D. Katholieke Universiteit Leuven
Department of Surgery Leuven, Belgium
Duke University Medical Center
Durham, North Carolina Alexei Vazquez, Ph.D.
The Simons Center for Systems Biology
Petra Levine Roulhac, Ph.D. Institute for Advanced Study
Department of Neurobiology Princeton, New Jersey
Duke University Medical Center
Durham, North Carolina Maria R. Warren, Ph.D.
UNC Duke Proteomics Center
Rukhsana Sultana, Ph.D. Program in Molecular Biology and
Department of Chemistry Biotechnology
Center of Membrane Sciences and University of North Carolina at
Sanders-Brown Center on Aging Chapel Hill
University of Kentucky Chapel Hill, North Carolina
Lexington, Kentucky
Leonard E. White, Ph.D.
Mao Tanabe, Ph.D. Department of Community and Family
Human Genome Center Medicine
Institute of Medical Science Doctor of Physical Therapy Division
University of Tokyo Department of Neurobiology
Tokyo, Japan Center for the Study of Aging and
Human Development
Duke University Medical Center
Durham, North Carolina
 1 Neuroproteomics
Oscar Alzate

Contents
1.1 Introduction.......................................................................................................1
1.2 Neuroproteomics...............................................................................................2
1.3 Experimental Techniques Currently Used in Neuroproteomics.......................3
1.3.1 Mass Spectrometry ...............................................................................3
1.3.2 Two-Dimensional Polyacrylamide Gel Electrophoresis........................4
1.3.3 Two-Dimensional Difference Gel Electrophoresis................................4
1.3.4 Liquid Chromatography........................................................................5
1.3.5 Protein Arrays........................................................................................5
1.3.6 Immunoblot...........................................................................................6
1.3.7 Analytical Ultracentrifugation..............................................................6
1.3.8 Surface Plasmon Resonance..................................................................6
1.3.9 Circular Dichroism................................................................................7
1.4 Challenges.........................................................................................................7
1.5 Organization of Book........................................................................................9
1.6 Final Remarks.................................................................................................. 10
References................................................................................................................. 11

1.1 Introduction
For the past several years, a large group of collaborators has been working together
toward understanding key biological problems related to brain function, brain struc-
ture, and the complexity of the nervous system. Problems such as the structure and
the function of pre- and post-synaptic densities, the sets of proteins that are regulated
by mental processes such as learning and memory formation, the protein networks
affected by apoE genotypes in Alzheimer’s disease patients, and the structure of
synaptic protein complexes in animal models of epilepsy, just to mention a few, have
been under scrutiny in these studies. To tackle biological problems using neuropro-
teomics, we have learned that multiple experimental approaches need to be imple-
mented. Techniques such as 2D-DIGE, mass spectrometry (MS), MS-based tissue
imaging, protein arrays, surface plasmon resonance (SPR), protein interaction net-
work analysis, multidimensional liquid chromatography, and many others are now in
daily use. This book is intended to provide the reader with an introduction to some
of the techniques that are most commonly used in neuroproteomics, and includes
some examples of how such techniques are used to understand biological processes.
A general overview of these techniques and their scope is discussed in this chapter.

1
2 Neuroproteomics

Modern biotechnology is enjoying an explosion of new systematic approaches for

the study of biomolecules and their interactions. The advent of genomics, proteom-
ics, metabolomics, peptidomics, lipidomics, glycomics, and all the other “omics” is
providing a vast amount of information that is enriching our knowledge of biological
systems. In this book, a group of experts in neuroproteomics and its applications pres-
ent the concept that understanding the dynamics of the proteome of a complex bio-
logical system requires the integration of many different experimental approaches.

1.2 Neuroproteomics
To define neuroproteomics we must start by understanding the term proteomics.
Although there are many definitions of proteomics, what we mean here by proteom-
ics is the study of a proteome (1), and a proteome is the complete set of proteins of an
organ or an organism at a given time and under specific physiological conditions. A
proteome is complex and refers to much more than the mere identification of the pro-
teins in the set. In any given proteome, proteins may interact with a certain number
of other proteins (or other molecules), determining how the protein functions as part
of the whole system. In addition, protein structure and/or function can be altered by
changes in the environment, including factors such as temperature, ionic strength,
pH, levels of oxidants or anti-oxidants, etc. (2). The study of the proteome should
provide information about all of these factors. Proteomics may start by elucidating
the “proteome” at a specific time, but it should also determine the “dynamics” of this
proteome under all the possible factors that affect the organ or organism. Thus pro-
teomics, by its very nature, is faced with a huge task that requires the collaboration
of multiple disciplines including physics, chemistry, biology, and bioinformatics.
Neuroproteomics is the sub-field of proteomics dedicated to answering these
same questions about the organs, tissues, and cells that make up the nervous sys-
tem (3–12). In neuroproteomics, our goals are (i) to identify all the proteins of a
given tissue, cell type, or organelle under specific conditions at a specific time; (ii)
to identify the post-translational modifications in all the proteins at that time and
under these conditions; (iii) to determine how this proteome changes as a function
of time (age), environmental changes, genetic factors, and with disease; and (iv) to
determine how these changes affect the organism as a whole. At the present time,
with existing technologies, the current knowledge of protein structure and func-
tion, our current knowledge about all possible protein–protein and protein–other
molecule interactions, and the financial resources available, the full achievement
of all of these goals is not possible. It is possible to find a “partial set” of proteins
from a given tissue, or to determine how a subset of these proteins changes under the
influence of some external factors such as a certain drug, etc. In fact, the complete
definition of a proteome as presented above has not yet been determined for any
organ or any tissue—not even for a single cell. This is what makes proteomics such
an interesting field—despite so much that has been accomplished we realize that
there is so much more to do.
In neuroproteomics the different pieces of the nervous system are “fragmented” so
that the dynamics of each given sub-proteome can be better understood. Just to men-
tion some examples (and this is not intended to be an exhaustive list), neuroproteomics
Neuroproteomics 3

works at solving the proteome of single neurons or astrocytes grown in cell cultures
or from primary brain cells isolated from tissues under several conditions (13–16);
at identifying a set of proteins characterizing a brain tumor (17–20); or at determin-
ing the set of proteins making up post-synaptic or pre-synaptic densities (21–27).
It is also common to try to solve a specific sub-proteome such as the heat-shock
response proteome (28–32), or the proteome responding to oxidative stress (33–37).
From these examples, it can be seen that specific groups of proteins are targeted for
analysis in a way that eventually will lead to solving a single proteome, and pos-
sibly being able to determine the dynamics of this proteome. The final goal will be
to be able to predict “how” the proteome will evolve when influenced by specific
conditions and to use this information to design methods that will modulate the
evolution of the proteome. This is the ultimate dream for rational drug design, and
molecular manipulation. The accumulation of huge amounts of data all around the
world requires the advent of better information systems that will permit this “global”
understanding of the dynamics of systems proteomics.
To accomplish the goals described above, proteomics requires the conjunction
of many disciplines and techniques. In this book, we describe some of these tech-
niques and give examples of several applications. A short description of the current
approaches used in neuroproteomics follows next. For a more detailed description of
some these techniques and their applications to proteomics, the reader is referred to
specific chapters in this volume and to the literature cited therein.

1.3 Experimental Techniques Currently

Used in Neuroproteomics
1.3.1 Mass Spectrometry
Mass spectrometry (MS) is the workhorse of proteomics. This technique offers tre-
mendous power in terms of sensitivity to detect either digested peptides or intact pro-
teins (38,39). Current developments allow targeting specific protein modifications
such as phosphorylation, oxidation, ubiquitination, and others (7,40,41), albeit with
varying degrees of difficulty and success. Unlike other approaches, such as x-ray dif-
fraction that require intact proteins either in crystal form or in solution, respectively,
MS requires that peptides or proteins be studied as ions in the gas phase.
As described in Chapter 5, matrix-assisted laser desorption/ionization (MALDI)
and electro-spray ionization (ESI) are the most common protein and peptide ion-
ization techniques used in MS-based proteomics. The availability of instrumenta-
tion and computer programs, together with the availability of protein databases
that can be used for automated comparisons with the experimentally derived–MS
and MS/MS (gas-phase sequencing) data, makes this the leading technique for
protein identification and characterization. In principle, hundreds to thousands of
proteins can be identified in a single sample using liquid chromatography (LC)
separation coupled on-line with these ionization techniques (LC-MALDI and/or
LC/MS/MS). The major obstacles that we face in applying MS to neuroproteom-
ics are limitations in sample availability, which plagues most neuroproteomics
experiments.
4 Neuroproteomics

A recent application of MS to neuroproteomics research is the molecular imag-

ing of brain tissues by MALDI, as described in Chapter 7. This approach, although
it has advanced tremendously under the leadership of Dr. Richard Caprioli, is still
in its infancy and its applications to neuroproteomics will be mainly for functional
analysis of subsets of proteins and for understanding the onset and progression of
neurological diseases. A great advantage of MALDI-based tissue imaging is the pos-
sibility of creating three-dimensional molecular images of the brain, or brain areas,
and then using these images to determine the dynamic evolution of sub-proteomes.
Mass spectrometry is also widely used to characterize protein post-translational
modifications (see Chapter 6). Trying to discover protein phosphorylation (42–44),
ubiqutination (24), palmitoylation, (45,46), oxidation (see Chapter 10) (33,47–51), and
other post-translational modifications (PTMs), and their roles in neurophysiology is
a challenging aspect of neuroproteomics. Chapter 6 describes mass spectrometry–
based approaches to characterize these and other PTMs relevant to brain function.

1.3.2 Two-Dimensional Polyacrylamide Gel Electrophoresis

Two-dimensional acrylamide-based gel electrophoresis is a powerful technique that
represents a tremendous resource for proteomics studies (see Chapters 3 and 4). Two-
dimensional polyacrylamide gel electrophoresis (2D-PAGE) allows the separation
of hundreds to thousands of proteins in a single experiment (40,52,53). Separated
proteins may be identified by mass spectrometry, or some potential modifications
may be analyzed “in” the gel. For example, some phosphorylated proteins can be
identified by staining with phospho-specific stains (see Chapter 4) (32) or phospho-
Ser/Thr-specific antibodies; oxidized proteins may be identified by Western blotting
(see Chapter 10) (33,37,54,55). 2D-PAGE is an extremely useful technique that offers
substantial advantages for quick and accurate protein separation and analysis, as
well as providing an extremely useful way of “visualizing” a complex sample. Both
1D- and 2D-PAGE can be combined with LC/MS-based separation of gel extracts
(“geLC”) to provide an additional dimension of separation for complex mixtures.

1.3.3 Two-Dimensional Difference Gel Electrophoresis

Differential-display proteomics (comparative proteomics) can be applied using mul-
tidimensional liquid chromatography or 2D-gel-based electrophoresis. Differential-
display proteomics is very powerful because it offers the advantage of comparing
several samples in a single experiment. The samples to be compared may include a
control (normal) versus an experimental (disease) sample (see Chapter 4) (6,53,56–
59). The experimental sample is selected such that it reflects the effects of a specific
condition including the effects of age, a drug, a gene, pH, oxidative stress, heat, etc.
The most common approach for differential display is 2D-difference gel electro-
phoresis (2D-DIGE). This technique permits comparing thousands of fluorescently
labeled proteins in a single experiment (see Chapter 4). With this technique, it is
possible to determine changes in protein expression, as well as potential post‑trans-
lational modifications (32,60,61). In addition, different types of fluorophores can be
used to probe specific properties of a protein. These may include cysteine, tyrosine,
Neuroproteomics 5

lysine, and histidine modifications and, virtually, any modification for which a spe-
cific fluorophore can be found. 2D-DIGE can be used to study post-translational
modifications including ubiquitination, phosphorylation, oxidation, and palmitoyla-
tion, among other modifications. As with any other technique, 2D-DIGE has limita-
tions and advantages, and it is commonly understood in proteomics laboratories that
a single technique will not suffice to answer all possible questions about a particular
proteome. Proteins isolated and analyzed by 2D-DIGE can be identified and char-
acterized by MS, or combined with immunoblotting for the analysis of specific sub-
proteomes (3,21,62).

1.3.4 Liquid Chromatography
A workhorse technique that has experienced tremendous advances, liquid chro-
matography (LC) offers the advantage of separating proteins in a liquid phase (see
Chapter 3) (6,63–65). Advances in all the technical aspects associated with LC make
this a good complement for any proteomics laboratory. Proteins isolated by LC may
be identified and characterized by MS, or they can be run on a 1D- or 2D-PAGE gel
for further comparisons (6,63,65,66). In neuroproteomics, the major challenge for
LC or LC/MS (see Chapters 3 and 5) is the scarce amount of samples for analysis.
Large columns, with large volumes, are therefore not recommended; instead, nano-
scale separations, micro-fluidic devices and affinity chromatography with specific
antibodies or other molecules to enrich the target molecules may be the methods of
choice (67,68). A major disadvantage of these nanoscale separations is the restrictions
placed on flow rates as well as the challenges of reducing dead volumes. For on-line
LC/MS, there are also restrictions on the selection of buffers and detergents. LC may
be combined with differential display proteomics or extended to multidimensional
approaches to provide a wider range of proteomics applications (see Chapter 3).

1.3.5 Protein Arrays
Protein arrays are designed to identify protein interactions using a solid surface to
capture the proteins of interest, or to characterize a property of a protein of inter-
est (16,41,53,69). This technique can be combined with other approaches such as
2D-DIGE and MS to identify specific proteins that interact with antibodies, peptides,
or other suitable molecules properly attached to solid surfaces. In neuroproteom-
ics, we use protein arrays to explore the changes in protein concentration, protein
modifications, and protein–protein interactions in nervous systems such as neurons,
axons, post-synaptic densities, etc. In this technique, protein lysates are incubated
with arrays of antibodies against the protein of interest. (For a detailed discussion
of protein arrays, protocols, and application see ref. 69.) The advantage of the arrays
is that specific groups of proteins can be targeted for analysis—for instance, mito-
chondrial proteins, synaptic proteins, or membrane proteins. The molecules immobi-
lized to the solid surface to produce the arrays can be specific for phosphorylated or
oxidized proteins. In principle, this approach is comparable to multiplexed Western
blots except instead of associating proteins one by one, molecular associations are
made against a large number of proteins in a single experiment (69).
6 Neuroproteomics

1.3.6 Immunoblot
Western blots (WBs) constitute a unique approach that allows identification of dis-
crete proteins using targeted antibodies against specific proteins (70,71). Widely
used in biological research, WB allows characterization of discrete changes in pro-
tein expression, and in combination with 2D gels, also allows one to detect changes
in protein isoforms, or post-translational modifications (32,37,52,72). The current
use of multiplexed WB using pre-labeled fluorescent antibodies permits quantitative
characterization of multiple protein changes in a single experiment. Extensive use of
WB can be used to determine protein changes in specific systems such as post-syn-
aptic densities (73), mitochondria, etc. This approach is equivalent to a single pro-
tein array, or to a low-resolution, non-quantitative molecular image as obtained by
MALDI-based tissue imaging (see Chapter 7). Several factors limit the application
of immunoblotting for proteomics. These include the low specificity of many anti-
bodies, and the lack of availability of pure antibodies. Moreover, antibodies simply
do not exist for some proteins. Currently an antibody proteome project is under way
that will provide a valuable tool for neuroproteomics research (http://www.hupo.org/
research/hai/) (74,75).

1.3.7 Analytical Ultracentrifugation
Analytical ultracentrifugation (AUC) offers a fast and reliable method for the deter-
mination of the molecular weight of a protein, and its hydrodynamic and ther-
modynamic properties (76,77). AUC is based on the thermodynamic analysis of
sedimentation equilibrium, and may be used to determine sample purity, integrity
of the structure, and degree of aggregation. The molecular weight determined by
AUC is that of the native state of the protein, as opposed to the unfolded state as
determined by gel electrophoresis, or in the gas state as determined by MS. This
technique can be used for the study of small molecules (several hundreds of daltons
of molecular weight such as small peptides) to multi-million-Da assemblies such as
viruses or multicomplex proteins, and organelles (76–80). AUC can be applied to
small samples in small volumes as are commonly found in neuroproteomics studies.
Sedimentation equilibrium can be used to determine the molecular weights of pro-
tein complexes as they exist in solution, including the determination of aggregation
states, and to study protein–protein interactions and protein interactions with small
molecules (80–82).

1.3.8 Surface Plasmon Resonance

Surface plasmon resonance (SPR) is a powerful technique to determine protein–pro-
tein interactions in solution (83–87). This technique offers the incomparable ability
to determine association (Ka) and dissociation (Kd) constants between two ligands
(84,85). A combination of SPR and mass spectrometry allows the identification of
protein interaction networks because the proteins bound to a ligand or a group of
ligands can be identified by MS (86,87). This technique offers precise quantitative
parameters that cannot be determined with protein arrays alone.
Neuroproteomics 7

1.3.9 Circular Dichroism
This technique is largely ignored in the proteomics field, mostly because high-
throughput approaches have not been developed. Circular dichroism (CD) spec-
troscopy offers the advantage of providing a fast and reliable screening for protein
secondary and tertiary conformations in solution (88). Many proteins associated
with neurological diseases including the amyloid β peptides, α-synuclein, and prion
proteins, for instance, form oligomeric conformations, which may be associated with
onset and progression of some diseases (see Chapter 9) (89–93). CD is a reliable
method for determining the folded conformations of these proteins. Implementing
high-throughput CD will provide a method to categorize these proteins and their
folded state. For now, neuroproteomics must use CD on discrete samples.

1.4 Challenges
Neuroproteomics faces many challenges (7,8,38,66,94). For many years neurosci-
entists have been trying to answer questions such as “What is conscience?” “What
are dreams made of?” “What are the physical substrates of memory and learning?”
“What are the differences between short-term and long-term memories?” and so on.
We are confident that neuroproteomics will offer a tool for neuroscientists working
on these and many other brain- and mind-associated questions. It is not our expec-
tation that proteomics alone will answer these questions, but instead it will be a
powerful tool that will help in the search for the right answers (7,8,66). At the pres-
ent time, being able to associate protein expression and protein modification with
electrophysiological data and with some of the superior functions of the brain will be
a good start. This is an area on which many research groups are working, and will
be a fantastic beginning of a bright future for neuroproteomics.
Among the many challenges, the one that we face every day and that needs to
be solved “up-front” so that data collection can be successful is sample prepara-
tion. The common presence of non-proteinaceous components such as nucleic acids,
lipids, carbohydrates, and other biomolecules can affect the outcome of a proteom-
ics analysis (see Chapters 3 and 4 for discussions on sample preparation). Many
of these “contaminants” may actually be part of a functional proteome. Some of
these biomolecules may be part of modulatory mechanisms for enzymatic reactions,
including DNA-, RNA-, lipid-, glycolipid-, and carbohydrate–protein interactions.
Therefore, elucidating what is a contaminant and what is part of modulatory mecha-
nisms in the cell or organelle is a challenge that needs to be addressed.
Another problem that needs to be addressed is the availability of enough sample
for proteomics analysis. For example, when working with post-synaptic densities
(PSDs) it is possible to obtain low (tens of micrograms) amounts of proteins. In
many cases, these protein lysates have to be cleaned for reliable analysis. During
this cleaning process, the amount of protein may decrease between 10% and 40%.
Assuming that the PSD contains several hundreds of proteins at any given time,
this means that the sample contains on average only a few tens of nanograms of
most proteins. Even this “best case” scenario represents the expression levels of the
high abundance proteins. Unfortunately, protein identification and characterization
8 Neuroproteomics

by mass spectrometry require at least 20 to 30 ng in most cases. This means that

many proteins—and proteins that are usually less abundant—will not be detected,
and possibly may not be identified, or may be identified but with insufficient peptide
coverage for modification site analysis. In most situations these problems need to be
addressed on a case-by-case basis, and individual solutions may be found depend-
ing on the availability of animal models or tissues to solve particular situations. We
should keep in mind that our goal is not to create catalogs of proteins; we are trying
to tackle biological questions relevant to the nervous system. It is also important to
keep in mind that in neuroproteomics we are interested in finding functional molecu-
lar pathways and not just simply trying to create a catalog of proteins in a tissue or
an organelle. The major goal is to determine the dynamics of the proteome because
“snapshots” alone do not provide enough valuable information about the changes
occurring within the system and the other systems associated with it.
Post-translational modifications (PTMs) are another big challenge that neurosci-
entists need to address in their quest to understand the molecular mechanisms asso-
ciated with brain functions. Some PTMs have been extensively studied, as is the case
with phosphorylation, oxidation, and ubiquitination. The role of these PTMs in some
cases has been addressed with optimistic results. Other PTMs such as sumoylation,
palmitoylation, and methylation have been less studied but are equally important.
However, there are a lot more PTMs than those mentioned above, and the complete
picture of their roles in brain functions needs to be addressed (52,95–100). The tech-
niques described in this book (see Chapters 3, 4, 6, and 7) provide some experimen-
tal approaches to address some of the questions associated with functional PTMs,
but this is just the beginning. A lot more work needs to be done.
The field of neuroproteomics requires a lot more work and a lot of dedication to
uncover the protein interaction networks (PINs) (see Chapters 8 and 9) (26,101–108).
Currently, PINs are evaluated using genomics data, or by using discrete groups of
proteins. However, a global view of the PINs associated with normal- and diseased-
brain functions is still not available. Along with the identification of the PINs, it is
very important to develop methodologies for their validation, and it is even more
important to develop tools that will allow researchers to predict the function and the
evolution of these networks under normal and disease conditions, under the effect of
aging, or due to environmental factors.
A major push for neuroproteomics has been the as-yet-unfulfilled dream of find-
ing “protein”-based biomarkers for neurodegenerative diseases. Thus far, proteomics
has been unable to deliver on this dream. Why? There are many reasons why we still
do not have hundreds of true protein biomarkers. We know a lot about individual
proteins, but all of the aspects concerning a proteome (as described above) are not
yet known even for a single proteome, and we know even less about the dynamics of
proteomes. It will be possible to find true and very useful biomarkers, but this will
have to wait a little longer while new technologies and—more importantly—new
concepts are developed for determining a whole proteome and how it changes with
time and environmental factors.
Neuroproteomics 9

1.5 Organization of Book

The book contains two major parts: the first part describes basic concepts for those
principles used in neuroproteomics (Chapters 2 to 8) (Figure 1.1), while the second
part is dedicated to illustrating a few examples showing how scientists are using
basic principles and techniques to understand molecular mechanisms of neurobio-
logical processes (Chapters 9 to 15) (Figure 1.1). These examples have been selected
while keeping in mind the ideas described above, i.e., that neuroproteomics is a set
of tools to be used for understanding fundamental neurobiological questions, and not
simply to create a catalog of nervous system-associated proteins.
As described earlier, one of the major challenges for neuroproteomics is sample
preparation. Chapters 2, 3, and 4 discuss experimental considerations, methods, and
concepts associated with sample preparation. Chapter 2 describes the experimen-
tal considerations for brain tissue collection and storage. Postmortem tissue is of
major interest for research targeting molecular mechanisms of neurodegenerative
Functional Analyses
Genomics
Tissue
Imaging
Prot-Prot
Sample Protein Protein Protein Interactions
Preparation Fractionation Identification Characterization
Prot-D/RNA
Interactions
Bio Markers
Tissue PTMs
2D-HPLC Prot-Lipid/Sugar Prognosis and
Dissection Protein
Interactions Diagnostics
Arrays
Mass
S.P.R.
Laser Capture Spectrometry
2D-GE
Microdissection
Mass 2D-DIGE Mass Spec
Spectrometry
Centrifugation 2D-DIGE
C.D. siRNA 2D-DIGE

Protein
Ab- Identification
Arrays

Data Mining

Chapters Chapters Chapters Chapters Chapters

2, 3, 4 3, 4 6 4, 6, 7, 9 8–15

Figure 1.1 Neuroproteomics requires many tools of modern biotechnology. As indicated

in the upper part of this figure, we are concerned with sample preparation, protein fraction-
ation, protein identification, and protein characterization. Then, we use a combination of
techniques such as mass spectrometry, protein arrays, 2D-DIGE, CD, gene silencing, ultra-
centrifugation, immunoprecipitation, and surface plasmon resonance to elucidate protein
function, protein–protein, protein–R/DNA, protein–lipid, and protein–carbohydrate interac-
tions. Using these data in the context of all the data available from public repositories, it is
possible to propose functional protein interaction networks, which may be validated by using
protein arrays and gene silencing followed by 2D-DIGE and mass spectrometry. The final
goal will be the possibility of predicting how a protein network would evolve and how it will
react under different experimental conditions. This will result in developing prognosis and
diagnosis techniques for neurological diseases, and will allow us to create molecular models
for neurological processes.
10 Neuroproteomics

diseases such as Alzheimer’s and Parkinson’s diseases. Proper collection, storage,

and manipulation of postmortem tissue are very important for reproducible and reli-
able proteomics analysis. The Bryan Brain Bank at the Department of Medicine and
Neurology at Duke University has been banking brain tissues for several years. This
bank specializes in brain specimens for neurodegenerative diseases, especially for
Alzheimer’s disease. Chapter 3 describes modern approaches for multidimensional
separations for neuroproteomics. These approaches are mainly based on liquid chro-
matography and are appropriate for every proteomics method. Chapter 4 is devoted
to differential gel electrophoresis (DIGE)-based proteomics. Methods required to
prepare proteins for DIGE are described in this chapter, along with experimental
considerations for a successful DIGE-based experiment. A discussion of DIGE
applications to neuroproteomics is presented in Chapter 4.
The technique most widely used for proteomics is mass spectrometry (MS); three
chapters are dedicated to mass spectrometry. Chapter 5 introduces the major con-
cepts of MS for proteomics analysis, including sources, detectors, and analyzers.
Chapter 6 presents major applications of mass spectrometry for the analysis of post-
translational modifications of proteins. A new application of MS is MALDI-based
tissue imaging. This technique has the potential to become one of the more useful
techniques for neuroproteomics. Chapter 7 describes the concepts, techniques, and
major applications of MALDI-based tissue imaging. Chapter 8 introduces the con-
cept of protein interaction networks, a new and rapidly evolving field based on the
theory of graphs that connects the elements of any group of proteins to other pro-
teins in the system, and how the inter- and intra-connectivity determines the overall
behavior of the system (101,104–107).
The second part of the book starts with an example of the identification of a
protein interaction network in neurodegeneration (Chapter 9). Chapter 10 analyzes a
large amount of data for studies of oxidized proteins in neurodegenerative diseases.
This chapter describes the use of 2D gels, mass spectrometry, and 2D Western blots
to tackle analysis of protein post-translational modifications. Chapters 11 and 12
present the use of neuroproteomics to study specific aspects of molecular mecha-
nisms in the visual system. Chapter 13 discusses networks of genes associated with
the auditory system, and it is accompanied by a neuroproteomics approach (Chapter
14) analyzing specific protein pathways associated with singing regulation in song-
birds. The last chapter, Chapter 15, presents several neuroproteomics approaches to
understand molecular mechanisms associated with nerve regeneration.

1.6 Final Remarks

This volume is intended as a general introduction to the concepts, techniques, and
applications of neuroproteomics. As presented in Figure 1.1, for neuroproteomics
studies we have to utilize multiple techniques for sample preparation, protein frac-
tionation, and protein identification. The combination of these approaches results in
the creation of extensive catalogs of proteins associated with a tissue or a system;
however, these catalogs are useless unless we can identify the function for each pro-
tein, the network to which each protein belongs, and the dynamics of the network
under experimental conditions. Facing these challenges requires the combination of
Neuroproteomics 11

many approaches, some of which have matured enough to be used in proteomics,

and some which are new and still under development. Combining mass spectrometry
with surface plasmon resonance, gene silencing, 2D-DIGE, and protein arrays will
help to discern these protein interaction networks and their dynamics.
It should be mentioned here that it is not possible to cover all aspects of neu-
roproteomics in a single volume. The field is growing so fast that it is not even
possible to review all the available literature. There are thousands of scientists
on all continents contributing to the many different fields of neuroproteomics,
including both technical developments and specific applications. In the present
volume, we tried to cover us much as possible of the current literature available,
and give specific examples that demonstrate exactly what neuroproteomics is,
and how and why it is used. I would like to apologize to all of those researchers
whose publications were not cited, which is not a disregard for their work, but
rather the result of how fast this field is growing, and lack of space in this single
volume. Also, we expect that the future will bring applications of techniques such
as x-ray crystallography, NMR, and EPR (electron paramagnetic resonance) to
neuroproteomics.
Finally I want to thank all the authors who have contributed to this volume. It has
been a difficult task that started more than five years ago, when we started design-
ing experiments, preparing samples, collecting data, and trying to validate as much
as possible the results obtained by proteomics approaches. Since then, the field has
grown considerably and today it is almost impossible to know everything being done
in neuroproteomics and its applications. This makes the efforts of all of the authors
more valuable as they have tried to give an up-to-date view of particular aspects of
this rapidly developing field.

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 2 Banking Tissue for
Neurodegenerative
Research
John F. Ervin

Contents
2.1 Introduction..................................................................................................... 17
2.2 Tissue Procurement and Storage..................................................................... 18
2.3 Other Considerations....................................................................................... 22
References................................................................................................................. 23

2.1 Introduction
Human brain banking has become an essential part of the research landscape
in neurodegenerative disorders and neurobiology. The demand for high quality
banked tissue has been on a steady rise for quite some time. Advanced research
studies, including proteomics, metabolomics, m-RNA micro arrays, and genom-
ics, are fast becoming the standard in neuroscience investigations. Since many
investigators study human diseases or biological processes, it is therefore not a
surprise that human tissue is in high demand to verify findings from animal mod-
els of disease. Many leading neuroscientists are focusing a large component of
their research on techniques that require the collection of the highest quality of
human brain tissue. The Kathleen Price Bryan Brain Bank (KPBBB; http://adrc.
mc.duke.edu/BB.htm) at Duke University Medical Center (DUMC) in Durham,
North Carolina, has over 20 years of experience with this process (1). Successful
human brain banking requires not only attention to the users’ needs for the high-
est quality of tissue, but it is also imperative for brain bankers to ensure that the
donor’s wishes are honored. There have been great strides on a national level to
facilitate the availability and distribution of these resources to the ever-growing
demand in the neuroscience community. The National Institute on Aging (NIA)
through the National Alzheimer’s Coordinating Center (NACC) has created an
infrastructure and informatics network to support collaboration among the indi-
vidual NIA-funded Alzheimer’s Disease Centers (ADCs) and to serve as a resource
for the neuroscience research community. The banked tissue thus obtained is an
invaluable resource available to qualified researchers. This chapter describes gen-

17
18 Neuroproteomics

eral concepts concerning proper acquisition, storage, and distribution of brain tis-
sue for neurodegenerative research.

2.2 Tissue Procurement and Storage

Although each ADC is required to report specific neuropathological data concern-
ing the tissue collected, each brain bank often varies in its procedures for procuring
its tissue for research. A protocol has not been established or accepted that instructs
how each bank should go about its task of tissue collection. Historically, each bank
may collect tissue according to different protocols in order to accommodate the spe-
cific needs of the investigators who most often use its tissue (2,3). The differences
in collection and storage protocols among ADC brain banks today are often driven
by the availability of resources and funding (4). Neuroscience investigators involved
with the NACC have become more organized in their efforts to amplify the useful-
ness of the individual brain banks that have collected tissue over the last several
decades. However, there has been some discussion about how modern brain banks
should be collecting and storing tissues in a way that provides high quality tissue for
a wider range of applications (5).
In addition to brain banks that are part of the NACC and focus on Alzheimer’s
disease (AD), there are other neurodegenerative investigative groups that have estab-
lished human brain banking resources. Parkinson’s, Huntington’s, amyotrophic lat-
eral sclerosis (ALS), and Creutzfeldt–Jakob disease (CJD) are also areas of research
that need human tissue to study (6,7). Although a majority of brain banks focus on
neurodegenerative research, there are several banks collecting tissue from individu-
als with psychiatric disorders, brain tumors, developmental anomalies and malforma-
tions, and brain tissue from acquired immunodeficiency syndrome (AIDS) patients
who are infected with human immunodeficiency virus (HIV) (8,9). Each of these
types of brain banks implements different protocols with a focus on collecting the
regions most affected in each disease process while emphasizing any special safety
and banking precautions that are needed for the inherent risks working with infec-
tious material (10,11). Regardless of the type of tissue banked, anyone who works
with fresh human tissue should take the necessary safety steps and follow universal
precautions for protection against the spread of infectious diseases.
The KPBBB has been collecting tissue since 1985 with an emphasis in Alzheimer’s
disease and other dementing illnesses. It takes a large amount of effort and coordina-
tion between a variety of committed personnel with highly specialized skills in order
to successfully retrieve, bank, and distribute quality tissue for today’s neuroscience
investigators. Having the proper facilities and trained personnel in place is not a
trivial matter when running a successful brain bank. The personnel and facilities
that are required often cross over different departments and disciplines within a
hospital. Coordinating and managing a working relationship among multiple entities
within a hospital setting can be a very laborious task. There are always funding and
cost issues that must be worked out by the collaborating parties. There are safety
concerns, a growing number of legal issues that must be fully addressed, and strict
adherence to the federal law is paramount when banking and distributing human tis-
sue. Without a knowledgeable liaison involving the different components, it would
Banking Tissue for Neurodegenerative Research 19

be very difficult to achieve a successful working relationship that is agreeable to all

those involved. The quality of tissue collected has a direct relationship to the effort
put forth by each and every person involved with coordinating the chain of events
that culminates with the banking of tissue that can be used for a variety of scientific
experiments.
The most useful specimens that are banked are derived from patients’ cases that
have been followed clinically and have a documented medical history that can be
used in conjunction with the neuropathological diagnoses. The process for procuring
the highest quality tissue often starts many years before the tissue is actually banked.
The neuropathology core is one component of a multidisciplinary team. The Joseph
and Kathleen Bryan Alzheimer’s Disease Research Center at Duke University con-
sists of a clinical core where patients are seen and treated at the clinic for memory
disorders. The clinical core facilitates the enrollment of their patients in a variety of
clinical studies as well as providing education for patients and family members about
the autopsy donation program.
Once a person decides to participate and become a tissue donor, the autopsy nurse
coordinator establishes a relationship with the donor and the donor’s family. Quite
often, many years may transpire between enrollment and tissue procurement. At
the time of enrollment, there are many legal documents that must be explained and
signed. There is a special relationship formed by the donor, the donor’s family, and
the coordinating nurse. These relationships are built on many discussions about the
donor and the family that can be quite personal in nature. The nurse coordinator is a
trusted source of information about what will happen to a loved one who has decided
to enroll in the autopsy program. These relationships are a fundamental part of a
successful donation program and they have translated into a high rate of participants
who are comfortable and willing to get follow-up exams to monitor their progress as
they age. It is important to remain in contact with the donor and his or her family,
so the nurse coordinator frequently makes house visits and may administer a neu-
ropsychological field test if the donor is not able to come to the clinic. However, in
the late stages of AD, the donor’s dementia may have progressed to the point that a
neuropsychological evaluation cannot be performed. As donors age or they enter an
advanced disease state, it becomes even more critical for the donor’s family to con-
tact the nurse coordinator so that when the last phase of the process begins everyone
is prepared.
A call from the next of kin or nursing facility usually notifies the nurse coordina-
tor that the donor has reached a point where he or she may expire in the next few
days. If possible, any perimortem observations are recorded and the autopsy nurse
coordinator will enter them into the medical record. Once the nurse coordinator
has been notified of a death several things must then be done in preparation for the
autopsy. Arrangements for the body to arrive at the hospital by a reliable transporta-
tion service will be required. Any available medical records and a signed consent for
autopsy must be given to the pathologist assigned to the case. The brain bank and
autopsy technicians are notified of the death as soon as possible in order to prepare
items for tissue collection and transport to the autopsy suite. If a donor lives too far
away to transport the body to DUMC for an autopsy to begin in a timely manner,
other arrangements can be made. Prior arrangements can be made for the tissue to be
20 Neuroproteomics

retrieved and shipped to the brain bank from almost anywhere in the United States.
The nurse coordinator can help a family get in contact with a facility and a neuro-
pathologist who are willing to help retrieve and ship the tissue to the brain bank.
The autopsy program nurse coordinator is a critical part of the team that facilitates a
smooth transition from donor enrollment to successful performance of the autopsy.
When the autopsy coordinator arranges for the transportation of the body to
DUMC, it is optimal for the body to be cooled down prior to arriving at the autopsy
suite. Depending on the expected delay to the hospital, the body may have been
refrigerated before delivery to the autopsy suite. The head would have been chilled
using a bag of wet ice in order to start cooling the brain. The autopsy technicians
will begin removing the brain as soon as the pathologist is finished with the external
exam. Ventricular cerebral spinal fluid (CSF) is taken using a large needle inserted
through the lateral side of the brain. The CSF is placed on ice for transport back to
the lab where it will be aliquoted into 1 mL vials and stored in a –80ºC freezer. Upon
removal from the cranium, the brain is weighed and grossly inspected. The specimen
is placed on wet ice in order to further cool the tissue. It is standard procedure for the
brain to be bisected through the corpus callosum, cerebellum, and brain stem. This
divides the brain into left and right hemispheres. One hemisphere of the brain will be
submerged in a tissue fixative such as 10% phosphate-buffered formalin for a mini-
mum of one week. The specimen can be dissected and paraffin-embedded after one
week of fixation. The embedded tissues are used for preparation of routine histologic
sections and a battery of immunohistological stains to detect plaques, tangles, Lewy
bodies or other inclusions. Before any tissue is used for research it must be evaluated
by a neuropathologist and the data catalogued in the research database. The fixed
tissue can remain submerged in formalin and kept at room temperature for long-term
storage. The other hemisphere may be fresh-frozen and kept free of any chemical
processes. The cerebellum and brain stem may be removed from the neocortex at the
level of the third nerve. This cut will expose the substantia nigra (Figure 2.1) so that
it can be removed from the brain stem and stored separately.

Figure 2.1 Substantia nigra: abnormal left; normal right.

Banking Tissue for Neurodegenerative Research 21

The fresh-frozen hemisphere will be coronally sliced 1- to 2-centimeters thick

from frontal pole to occipital pole. Cutting the hemisphere into coronal slices allows
access to internal structures that may be dissected out and stored. Commonly dis-
sected structures include the hippocampus (Figure 2.2A), amygdala, and basal gan-
glia (Figure 2.2B). Dissected pieces are placed in labeled aluminum foil and placed
into a container of liquid nitrogen to quickly freeze the tissue. The coronal slices are
placed in numbered freezer bags beginning with the frontal lobe and ending with the

Figure 2.2A  Hippocampus shown inside the small rectangles.

Figure 2.2B Amygdala within small rectangles; basal ganglia within the large
rectangles.
22 Neuroproteomics

occipital lobe. These bags are placed on wet ice for transportation back to the labora-
tory. The cerebellum and brain stem are placed in separate bags to be transported
and frozen with the coronal slices. The bags are frozen between chilled steel plates
in an ultra low –80ºC freezer. Long-term storage of frozen specimens at most banks
is in ultra low –80ºC freezers.
In advance of an autopsy, there are three things that must be considered. How
will the tissue be dissected, preserved, and stored? As discussed above, not all brain
banks support the same research interests. These differences will influence each of
the three components of the brain banking process. Each type of bank will need
to establish an autopsy protocol based on what is important for its research focus.
The KPBBB has predominantly focused on banking Alzheimer’s disease and aged-
matched control tissues. However, we also have several other types of neurodegen-
erative diseases represented in our collection. Each neurodegenerative disease has a
specific brain region that is most affected. These regions are what investigators most
often request from the bank for their research. For example, in Parkinson’s disease,
the substantia nigra is most often sought after. In Huntington’s disease, the basal
ganglia are of interest, specifically including the caudate nucleus. The hippocampus
is the most requested region associated with Alzheimer’s disease research. In addi-
tion to dissecting each of these areas, we also collect other structures including the
amygdala. We enroll normal controls, which would have the same regions retrieved
at the time of autopsy. Due to the availability of these tissues in our collection, we
have distributed tissue for a wide variety of studies over the years. No matter which
areas are chosen for special dissection, it is best to retrieve frozen tissue as consis-
tently as possible to maximize its usefulness in comparative studies.

2.3 Other Considerations
There are ongoing discussions in the neuroscience community as to what methods
a brain bank should employ in order to maximize the usefulness of its frozen tissue
(5,12). Some banks dissect different brain regions and also use a different method of
freezing. There are methods of freezing other than using liquid nitrogen or chilled
metal plates in a –80ºC freezer. Some banks use an isopentane/dry ice slushy mix-
ture, while others use only the vapor from the liquid nitrogen. Some banks cryopro-
tect the samples before freezing and some do not. Selecting tissue for comparative
studies has many variables that come into play when an investigator requests tissue
from a brain bank. The method of freezing can be an issue depending on the project.
We are asked to select disease cases with matching normal controls using gender,
age at death, apolipoprotein E (APOE) genotype, and specific neuropathy diagnosis.
In addition to these variables, most investigators insist on using the shortest post-
mortem interval (PMI) possible. All of these details are databased and can be que-
ried in order to find appropriate matches. However, the number of specimens that can
be selected for a project decreases with each variable. Tissue that is prepared using
similar methods by different neurodegenerative brain banks allows investigators to
request tissue from several brain collections.
The advancement of scientific techniques used in gene expression studies has
been driving the debate concerning the methods of banking diseased neurological
Banking Tissue for Neurodegenerative Research 23

tissue. There has been published research to show that biomolecule stability is highly
variable. These studies show that the stability of proteins and RNA transcripts is not
guaranteed using screening criteria such as PMI (13–15). PMI is the time it takes to
get the tissue in the freezer after death. Cooling the tissue as soon as possible seems
to be very beneficial in preserving intact RNA and proteins. However, it appears that
individual proteins and RNA transcripts may react differently under the same condi-
tions. These studies point to a lack of predictability of biomolecule integrity using
measurements such as PMI. There are some biomolecules that can withstand a wider
range of PMIs and some that cannot. It is expected that some biomolecules may not
be recoverable due to factors that promote degradation no matter how optimal the
conditions are during tissue retrieval. At this time it is prudent to evaluate the post-
mortem integrity of the molecules of interest before conclusions can be drawn about
the role of these molecules in the pathogenesis of any disorder.

References
1. Hulette, C. M., Welsh-Bohmer, K. A., Crain, B. et al. (1997) Rapid brain autopsy. The
Joseph and Kathleen Bryan Alzheimer’s Disease Research Center experience, Arch
Pathol Lab Med 121:615–8.
2. Cochran, E. J., Gostanian, O. M. and Mirra, S. S. (1995) Autopsy practices at CERAD
and Alzheimer disease center sites: A survey of neuropathologists, Alzheimer Dis Assoc
Disord 9:203–7.
3. Tourtellotte, W. W., Rosario, I. P., Conrad, A. and Syndulko, K. (1993) Human neuro-
specimen banking 1961–1992. The National Neurological Research Specimen Bank (a
donor program of pre- and post-mortem tissues and cerebrospinal fluid/blood; and a col-
lection of cryopreserved human neurological specimens for neuroscientists), J Neural
Transm Suppl 39:5–15.
4. Hulette, C. M. (2003) Brain banking in the United States, J Neuropathol Exp Neurol
62:715–22.
5. Vonsattel, J. P., Del Amaya, M. P. and Keller, C. E. (2008) Twenty-first century brain
banking. Processing brains for research: The Columbia University methods, Acta
Neuropathol 115:509–32.
6. Cruz-Sanchez, F. F., Moral, A., de Belleroche, J. and Rossi, M. L. (1993) Amyotrophic
lateral sclerosis brain banking: A proposal to standardize protocols and neuropathologi-
cal diagnostic criteria, J Neural Transm Suppl 39:215–22.
7. Reynolds, G. P. and Pearson, S. J. (1993) Neurochemical-clinical correlates in
Huntington’s disease—Applications of brain banking techniques, J Neural Transm
Suppl 39:207–14.
8. Haroutunian, V. and Pickett, J. (2007) Autism brain tissue banking, Brain Pathol
17:412–21.
9. Schmitt, A., Bauer, M., Heinsen, H. et al. (2007) How a neuropsychiatric brain bank
should be run: A consensus paper of Brainnet Europe II, J Neural Transm 114:527–37.
10. Bell, J. E. and Ironside, J. W. (1997) Principles and practice of ‘high risk’ brain banking,
Neuropathol Appl Neurobiol 23:281–8.
11. Morgello, S., Gelman, B. B., Kozlowski, P. B. et al. (2001) The National NeuroAIDS
Tissue Consortium: A new paradigm in brain banking with an emphasis on infectious
disease, Neuropathol Appl Neurobiol 27:326–35.
12. Webster, M. J. (2006) Tissue preparation and banking, Prog Brain Res 158:3–14.
24 Neuroproteomics

13. Crecelius, A., Gotz, A., Arzberger, T. et al. (2008) Assessing quantitative post-mor-
tem changes in the gray matter of the human frontal cortex proteome by 2-D DIGE,
Proteomics 8:1276–91.
14. Ervin, J. F., Heinzen, E. L., Cronin, K. D. et al. (2007) Postmortem delay has minimal
effect on brain RNA integrity, J Neuropathol Exp Neurol 66:1093–9.
15. Lipska, B. K., Deep-Soboslay, A., Weickert, C. S. et al. (2006) Critical factors in
gene expression in postmortem human brain: Focus on studies in schizophrenia, Biol
Psychiatry 60:650–8.
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It is a mood in which the bounds between romance and allegory fade
away; persons become symbols and symbols have breathed into
them the breath of life. The story and the truth it shadows are one.
The mood is common in poetry. Poets like Dante and Spenser and
Shelley from it have given us

Wise and lovely songs

Of fate and God and chance and chaos old,
And love.

There is a point where “dreams begin to feel the truth and stir of
day,” where the incidents of existence assume a dream-like
character, and where dreams become transparent symbols of reality.
There are moods in which our familiar world seems strange to us,
and we walk in it as on some bewildered shore.
In such moods to meet Hawthorne is a great experience. He is no
longer shy and aloof, but he opens to us his heart, and with friendly
zeal points out each object of interest—for in this border-land he is at
home.
 THE CRUELTY OF GOOD PEOPLE

T HE cruelty of bad people is easily explained. They are cruel because

they enjoy watching the pain of others. There are also the ignorant
and half-formed, to whom the word “inhumanity” applies literally. They
have not yet been really humanized. Before they can habitually yield to
feelings of compassion there is much to be done in developing their
higher natures. They must be urged to

Move upward, working out the beast,

And let the ape and tiger die.

The beast has a long start, and the ape and tiger die hard.
But this is only half the story. We are continually surprised at the cruelty
that is possible in those in whom there seems to be no tigerish survival. It
is intimately associated with the higher rather than with the lower part of
the nature. It is spiritual, rational, and moral. The cruelty of women and
priests is proverbial—and they are good women and good priests.
Listen to the talk in a drawing-room when some question involving the
fate of thousands is introduced. There is a strike or lock-out. It means that
the hostile parties are struggling on a narrow ledge between two
precipices. The workmen are trying to push the employers into the abyss
of bankruptcy; the employers are exerting every means in their power to
hurl their antagonists into the abyss of starvation. It is a battle to the
death, and in many a home pale-faced women are watching it with
despairing eyes. But what says my lady who likes to talk about current
events? It is evident when she begins to speak that she is not touched by
the tragedy of it all. Nero watching the burning of Rome could not assume
an air of more complete detachment. She talks as if it were nothing to her.
Or the talk turns to the affairs of state. Issues that involve the fate of
nations awake in her only a languid curiosity. The diplomacy of prudent
statesmen who are endeavoring to keep the peace strikes her as mere
dilly-dallying. She wants to see something doing. She enjoys a romantic
sensation, and urges on those who would give her this pleasure. Was
there ever a useless war without fair faces looking down upon it
approvingly—at least at the beginning?

I saw pale kings, and princes too,

Pale warriors, death pale were they all;
They cried, “La Belle Dame sans Merci
Hath thee in thrall.”

Yet she who in regard to the great affairs which involve millions may
appear as “La Belle Dame sans Merci” may be to all those whom she
knows a minister of purest kindness. It is only towards those whom she
does not know that she is pitiless.

* * * * * *
Philosophers are usually cruel in their judgments of the persons and
events of the passing day, and that is perhaps the reason why no nation
has been willing to take the hint from Plato and allow the philosophers to
rule. It would be too harsh a despotism. Flesh and blood could not endure
it. For the philosopher is concerned with general laws and is intolerant of
exceptions, while it is the quality of mercy to treat each person as in some
degree an exception. Fancy the misery that would be involved in the
attempt to level us all up to the cold heights of abstract virtue on which
Spinoza dwelt. One shudders to think of the calamity that would ensue
were all our lawmakers to be suddenly Hegelianized. All the attempts to
alleviate the hard conditions under which people are living would cease.
The energy that is now spent in trying to abolish abuses would then be
directed toward explaining them. What wailings would go up from earth’s
millions on the proclamation of the rule of unlimited Spencerianism! We
should look back with envy to the good old times of Nero and Tamerlane.
As the Inquisition handed its victims over to the secular arm and
disclaimed all further responsibility, so this new tyrant would hand over all
the unfit to the unobstructed working of natural law. No attention would be
paid to our sentimental preferences for particular persons. Those merciful
interferences which have been the contrivance of mankind for the
protection of weakness must be swept aside. The unfit must take the full
penalty justly visited on their unfitness. The moment we begin to
particularize we rebel. Pity revolts against a too cold philosophy.
It is needless to say that the theologians have often attained refinements
of cruelty unknown even to the most severely logical of the secular
philosophers. They have been able to distill out of the purest religious
affections a poison capable of producing in the sensitive soul unutterable
agony. Then they have watched the writhing of the victim with a cold
benevolence. The worst of it was that the benevolence was real in spite of
the fact that it froze all the fountains of natural pity.
Jonathan Edwards was not merely a good man in the ordinary sense. His
goodness rose into ideal heights. He had a genius for ethics as well as for
religion. He is still a teacher of teachers. But this wonderful man, who
must ever have a high place among the leaders and inspirers of mankind,
has an equally high place among the torturers of the spirit. To understand
the kind of pain which he inflicted we must not be content with the
threatenings of torment in sermons like that on “Sinners in the Hands of
an Angry God.” The pictorial imagery which now startles us was common
enough in his day. The torments of sinners was an ordinary theme;
Edwards added appreciably to the torments of the saints. His vivisection
of the human soul was without compunction. In the hearts and desires of
the innocent he discovered guilt for which there was no pardon. Every
resting-place for natural human affection was torn away, and when at last
from the clear heaven the love of God shone down in dazzling splendor, it
shone upon a desert.
The cruelty of it all is seen in its effects on minds naturally prone to
melancholy. Read the journal of a disciple of Edwards, David Brainerd,
and remember that for several generations that journal was esteemed a
proper book to put into the hands of youth. The editor of the Journal says,
“As an example of a mind tremulously apprehensive of sin, loathing it in
every form and for its own sake, avoiding even the appearance of evil,
rising above all terrestrial considerations, advancing rapidly in holiness,
and finding its only enjoyment in the glory of God, probably no similar
work in any language can furnish a parallel.” Poor Brainerd! Every step
along the heavenly way cost him a pang. He never could forget for more
than a few hours at a time that he was human, and to be human was to
be vile. The groans follow one another with monotonous iteration. He
loved God, but he felt his guilt in not loving him more. He was not only
afraid of hell, but of a heaven of which he was unworthy.
“I seem to be declining with respect to my life and warmth in divine things.
I deserve hell every day for not loving my Lord more.... I saw myself very
mean and vile, and wondered at those who showed me respect.”
We all feel that way sometimes, but to have the feelings set down day by
day for years at a time seems hardly profitable. We are relieved when
occasionally the editor summarizes the spiritual conflicts of a week or two
without going into details, as in the latter part of December, 1744. “The
next twelve days he was for the most part extremely dejected,
discouraged and distressed, and was evidently much under the power of
melancholy. There are from day to day most bitter complaints of
exceeding vileness, ignorance, and corruption; an amazing load of guilt,
unworthiness even to creep on God’s earth, everlasting uselessness,
fitness for nothing, etc., and sometimes expressions even of horror at the
thoughts of ever preaching again. But yet in this time of dejection he
speaks of several intervals of divine help and comfort.”
The pitiful thing about it all was that Brainerd’s distress arose not from the
consciousness of any particular shortcoming of his own, which after all
was finite. He was endeavoring to realize the meaning of that infinite guilt
which was his as a child of Adam. That guilt must be infinite because it
was a sin against infinite purity and power. When he had repented to the
very utmost of his ability, he was conscious that he had not repented
enough.
When he went to New Jersey as a missionary to the Indians, it was this
abnormal spiritual sensitiveness which he endeavored to impart to the
aboriginal mind.
He found it difficult to bring the Indians to that degree of spiritual anguish
which, in his view, was necessary to their salvation. He could make them
understand the meaning of actual transgression, but they were dull of
comprehension when he urged them to repent of original sin.
“Another difficulty,” he says, “which I am now upon, is that it is next to
impossible to bring them to a rational conviction that they are sinners by
nature, and that their hearts are corrupt and sinful, unless one can charge
them with some gross act of immorality such as the light of nature
condemns.”
One would suppose that the missionary might have found among his
untutored Indians enough actual transgressions to have brought to them a
conviction of sin and a desire for a better life. But no, that was not
enough, it would have fallen far short of what he had in mind. It would
have only convinced them that they were sinners individually considered,
and would not have overwhelmed them with the guilt of the race. So he hit
upon a device to turn their minds from the incidental trangressions of
mature life to the central fact that depravity was innate and universal.
“The method which I take to convince them that we are sinners by nature
is to lead them to an observation of their little children: how they will
appear in a rage, fight and strike their mothers before they are able to
speak or walk, while they are so young that they are incapable of learning
such practices.... As children have never learned these things, they must
have been in their natures; and consequently they must be allowed to be
by nature the children of wrath.”
It did not seem to occur to Brainerd that in thus setting the child in the
midst of them as an illustration of the kingdom of wrath he was not
imitating the method of Jesus. Even in his treatment of the sins of later life
there is something illustrative of the cruel system which dominated him.
“I then mention all the vices I know the Indians to be guilty of, and so
make use of these sinful streams to convince them that the fountain is
corrupt. This is the end for which I mention their wicked practices to them;
not because I expect to bring them to an effectual reformation merely by
inveighing against their immoralities, but hoping that they may hereby be
convinced of the corruption of their hearts, and awakened to a sense of
the depravity and misery of their fallen state.”
Brainerd had in mind a profound truth; every great moral awakening is
accompanied by pain. But he was not content with that which comes
naturally. All specific reformation in morals and manners was
subordinated to that which he conceived to be the essential thing,—that
they should feel to its full extent the misery of being human.

* * * * * *
In every readjustment of thought or advance in the manner of life there is
involved a vast amount of unescapable pain. There is also a great deal of
pain that is gratuitously inflicted. In the contest between the forces of
conservatism and progress it is difficult to say which side is more open to
the charge of cruelty.
In reading history our sympathies are usually with the bold innovator. He
stands alone against the world and proclaims an unpopular truth. He is
misunderstood, reviled, persecuted for righteousness’ sake. The
defenders of the old order are hard-hearted persecutors who hound him
to death.
But this is only half the story. A glimpse of the other side is given in the
very term we use. We speak of the defenders of the old order. We only
understand their feelings when we remember that they were really on the
defensive. The things they held most sacred were attacked by a ruthless
power which they could not understand. They flew to the rescue of
sanctuaries about to be violated. They often fought as those in mortal
agony, using blindly such weapons as came to their hands.
In “The Faerie Queene” Una, the fair symbol of Truth, wanders through
the forest protected by her lion. He is a good lion and faithful to his lady.

The lyon would not leave her desolate,

But with her went along, as a strong gard
Of her chast person, and a faythfull mate
Of her sad troubles and misfortunes hard:
Still when she slept, he kept both watch and ward;
And when she wakt, he wayted diligent
With humble service to her will prepard;
From her fayre eyes he took commandëment
And ever by her lookes conceived her intent.

That is the picture that comes to the adherent of the old order. The pure
virgin Truth walked unharmed, with her strong protector by her side. At
length a proud Paynim attacked the gentle lady. Then it was that

her fiers servant, full of kingly aw

And high disdaine, whenas his soveraine Dame
So rudely handled by her foe he saw,
With gaping jawes full greedy at him came,
And, ramping on his shield, did weene the same
Have reft away with his sharp rending clawes.

But it was a losing battle. The lion’s sudden fierceness was all in vain.

O then, too weake and feeble was the forse

Of salvage beast.

Now that her defender is slain, what is to become of Lady Truth?

Who now is left to keepe the forlorn maid

From raging spoile of lawless victor’s will?
The lover of the old order does not stop to ask whether the lion may not
have made a mistake, and whether the object of his attack may not have
been, instead of a proud Paynim, only a Christian knight who had
approached to ask his way. Nor does he feel pity for the pains inflicted by
the lion’s “sharp rending clawes.” He only cries, “Poor lion! Poor Lady
Truth!”
“But,” says the careful reader, “are you not getting away from your
subject? You proposed the question, ‘Why are good people so cruel?’ You
began with the conversation of excellent ladies in the drawing-room, and
now you have wandered off into faery land, and are talking about the Lady
Truth and the noble lion who died in her defense. I fear you are losing
your way.”
On the contrary, dear reader, I think, as the children say when they are
hunting the thimble, we are “getting warm.” We started out to find a cause
for the obliviousness of good people to the pain which they inflict on
others, and we have come into the region of allegory. Now, one of the
chief reasons why good people are cruel is that it is so easy for them to
allegorize.
In an allegory virtues and vices are personified. Each is complete in itself,
and when it once has been set going it follows a preordained course. It
does not grow into something else, and it is incapable of repentance or
improvement. In the morality plays a virtue is as virtuous and a vice as
vicious at the beginning as at the end. Spenser prefixes to “The Faerie
Queene” a prose explanation of the meaning of each important character.
“The first of the Knight of the Red-crosse, in whom I set forth Holynes; the
second of Sir Guyon, in whome I sette forth Temperance; the third of
Britomartis, a lady knight, in whom I picture Chastity.” Now, after this
explanation we are relieved of all those anxieties which beset us when we
watch creatures of flesh and blood setting out in the world to try their
souls. Everything is as much a matter of invariable law as the reactions of
chemical elements. The Knight of the Red-cross may appear to be
tempted, but he is really immune. He cannot fall from grace. From that
disaster he is protected by the definition. We have only to learn what the
word holiness means to know what he will do. As for Sir Guyon, when
once we learn that he is Temperance, we would trust him anywhere. For
such characters there is nothing possible but ultimate triumph over their
foes. And what of their foes? Being allegorical characters, they cannot be
reformed. There is nothing to do but to kill them without compunction, or if
we can catch them in the traps which they have set for others, and make
them suffer the torments they have themselves invented, so much the
better. We welcome the knight—

Who slayes the Gyaunt, wounds the Beast,

And strips Duessa quight.

We have no compunctions as we watch the administration of poetical

justice. Whatever happens to the false Duessa and to such miscreants as
Sansfoy and Sansjoy and Sansloy, we say that it serves them right.
If we can only hold fast to the allegorical clue, and be assured that he is
dealing with sins and not with persons, we can follow Dante through
purgatory without flinching. The moral always is a good one, and full of
suggestiveness.
But the moment we mistake an allegorical character for a person of flesh
and blood we get into trouble. Even the most perfect parable represents
only a certain phase of reality. When it is forced beyond its real intention
and taken literally it shocks our sense of humanity. It needs to be
interpreted by the same wise spirit that conceived it. We repeat the story
of the symbolic virgins who had forgotten to put oil in their lamps, or of the
servant who was too timid to put his master’s money out to usury. The
child asks, “Wasn’t it cruel of those wise virgins not to give the others just
a little of their oil? And after the door was shut and the foolish virgins
knew how foolish they were and were sorry, couldn’t the people inside
have opened the door just a little bit? And just because the servant was
afraid to go to the bank with the money, because it was so little, ought the
master to have been so hard with him as to say, ‘Cast ye the unprofitable
servant into the outer darkness; there shall be weeping and wailing and
gnashing of teeth?’ Why didn’t he give him another chance?”
Then the parent will explain that these are symbolic characters. Or
perhaps he may not try to explain, but change the subject and read a
story of real people like that of the prodigal son or the good Samaritan.
The child may be made to understand that while the door is always shut
against a sin, it is always open for the sinner who repents.
The sensitive child takes up the “Pilgrim’s Progress” and reads of the way
Christian went on his way to the heavenly city, meeting all kinds of people,
yet apparently without sympathy for most of them. “Why did he leave his
wife and little children in the City of Destruction and go off alone? If he
knew that the city was to be burned up, why didn’t he stay with them? He
doesn’t seem to care very much for what happens to people who are not
of his set.” So it seems to be. Mr. Hold-the-world, Mr. Money-love, and Mr.
Save-all walk along with him, and then they go off the path to look into a
silver-mine. Christian doesn’t take the trouble to find out what became of
them. Bunyan says coolly, “Whether they fell into the pit by looking over
the brink or whether they went down to dig, or whether they were
smothered by the damps that commonly arise, of these things I am not
certain; but this I observed, that they were never seen that way again.”
Christian goes on after the tragedy perfectly unconcerned, singing a
cheerful hymn. It was none of his business what happened to those who
wandered off the road. He is rather pleased than otherwise when Vain-
Confidence falls into the pit. When “the brisk young lad,” Ignorance, joins
him Christian converses with him only long enough to find out his name
and where he came from. Then instead of trying to improve him he leaves
him behind. Poor Ignorance trudges after, but he never can catch up.
All this is right in an allegory. Ignorance must be left behind, Vain-
Confidence must perish in the pit; from the City of Destruction we must
flee without waiting for others to follow. This is a very simple lesson in the
way of life. The next lesson is more difficult and it is quite different,—how
to treat ignorant and vainglorious and otherwise imperfect persons.
The first thing we have to remember is that they are persons, and that
persons are quite different from allegorical characters. Persons can
change their minds, they can repent and aspire after a better life, and
above all they have feelings,—which abstract virtues and vices do not
have. Does not the cruelty of the good chiefly arise from the fact that they
do not see all this?
In a preceding essay we have considered Hawthorne’s judgment on the
characters which he himself created. His most powerful story of sin and
retribution wears to his eyes “a stern and sombre aspect too much
ungladdened by the tender and familiar influences which soften almost
every scene of Nature and real life.” He was aware that he was depicting
not all of life, but only one aspect of it. He saw the characters of the
“Scarlet Letter,” as they saw themselves, “in a kind of typical illusion.” He
was fully aware that his treatment was symbolic rather than realistic. Real
life is infinitely more complex and therefore more full of possibilities of
good than any symbolic representation of it.
I do not think that good people are really as cruel at heart as one would
be led to think from their words, or even from their acts. I remember a
good professor of theology who was discoursing on the way in which the
Canaanites were destroyed in order that Israel might possess the land.
“Professor,” asked a literal-minded student, “why did the Lord create the
Canaanites, anyhow?”
“The Lord created the Canaanites,” answered the professor, “in order that
Israel might have something on which to whet his sword.”
The words were bloodthirsty enough; and yet had I been a Canaanite in
distress I should have made my way at once to the good professor’s
house. I am sure that the moment he saw me he would have taken me in
and ministered tenderly to my distresses and protected me from an
unkindly world. But I should have taken the precaution to let him see me
before he learned my name. A Canaanite in the abstract would be an
abomination to him, and I would have to take pains to make him
understand that I was a human being.
The word “cruel” is in its derivation akin to “crude;” it is that which is raw
and unripe. Like all other good things, righteousness at first is crude.
Crude righteousness takes no account of the difference between a sinner
and his sin; it hates both alike with a bitter hatred, and visits on each the
same condemnation. It is harsh and bitter. For all that it is a good thing,
this unripe fruit of righteousness. Give it time and sunshine, and it will
grow sweet and mellow.
The Riverside Press
Electrotyped and printed by H. O. Houghton & Co.
Cambridge, Mass., U. S. A.

Transcriber’s Notes:
Variations in spelling and hyphenation are retained.
Perceived typographical errors have been changed.
 *** END OF THE PROJECT GUTENBERG EBOOK THE
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