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Methods in
Molecular Biology 1862
Sarah-Maria Fendt
Sophia Y. Lunt Editors
Metabolic
Signaling
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:

http://www.springer.com/series/7651
Metabolic Signaling
Methods and Protocols
Edited by
Sarah-Maria Fendt
Laboratory of Cellular Metabolism and Metabolic Regulation, VIB-KU Leuven Center for Cancer
Biology, Leuven, Belgium
Sophia Y. Lunt
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA
Editors
Sarah-Maria Fendt Sophia Y. Lunt
Laboratory of Cellular Metabolism Department of Biochemistry
and Metabolic Regulation and Molecular Biology
VIB-KU Leuven Center Michigan State University
for Cancer Biology East Lansing, MI, USA
Leuven, Belgium
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-8768-9 ISBN 978-1-4939-8769-6 (eBook)
https://doi.org/10.1007/978-1-4939-8769-6
Library of Congress Control Number: 2018958961
© Springer Science+Business Media, LLC, part of Springer Nature 2019

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Preface
Metabolism is the biochemical reaction network that allows cells to convert nutrients into
small molecules, called metabolites. Through these metabolite conversions, essential com-
ponents needed for cell survival and proliferation are generated. For example, metabolite
conversions allow the production of ATP, the energy currency of cells, as well as the
production of amino acids, fatty acids, and nucleotides—the building blocks for proteins,
membranes, and DNA/RNA. Moreover, dynamics in metabolite concentrations facilitate
the crosstalk between cell signaling and the biochemical reaction network of metabolism.
This bidirectional crosstalk is necessary to enable the response of cells to stimuli such as
nutrient availability/limitations and growth factors. The central role of metabolism in
integrating directly or indirectly (via cell signaling) various external and internal stimuli
defines metabolic signaling.
In this book, we provide protocols to quantify metabolism (Chapters 1–10), to identify
metabolic crosstalk (Chapters 11–14), and to set up and develop tools and models to gain a
systems-level insight into metabolic signaling (Chapters 15–20). Quantifying metabolism is
essential to describe and understand metabolic signaling. Metabolite concentrations, meta-
bolic pathway activities, and metabolic fluxes constitute different functional readouts of
metabolism. Different methods are required to determine these readouts of metabolism.
Depending on the cellular system and the resolution level, variants of these methods should
be applied. The most global readout of metabolism are metabolite concentrations. Changes
in metabolite concentrations pinpoint the metabolite nodes within the biochemical reaction
network that respond to a certain stimuli or perturbations. In Chapter 1, Wang and
colleagues describe a protocol to determine the most suitable metabolomics method for
broad metabolite coverage, and in Chapter 2, Langerborg and colleagues provide a method
to specifically assess bioactive lipids. The activity of metabolic pathways and how they are
fueled by nutrients is an important aspect of understanding the metabolic requirements of
cells. In Chapters 3 and 4, Ogrodzinski et al. and van Gorsel et al. provide workflows for
applying nutrients labeled with a stable isotope of carbon to determine metabolic pathway
activity and nutrient contributions in vitro in adherent 2D and 3D spheroidal cell cultures,
respectively. In Chapters 5 and 6, Broekaert and Fendt and Pinnick et al. provide protocols
to extend the use of metabolites labeled with stable isotopes to analyze in vivo glucose and
lipid metabolism in mice and humans, respectively. Metabolic fluxes are the most quantita-
tive readout of metabolism, and several different and complementary approaches exist to
measure absolute fluxes in cultured cells. Bird et al. and Newman and Maddocks describe in
Chapters 7 and 8 methods to estimate ATP and amino acid synthesis rates, respectively, by
estimating the steady state flux with flux inhibition. Veys and colleagues apply in Chapter 9
radioactive tracers to determine fluxes in central carbon metabolism of endothelial cells,
while Nonnenmacher and colleagues estimate compartment-resolved metabolic fluxes in
Chapter 10.
Metabolic signaling describes the bidirectional crosstalk between external stimuli and
cell signaling pathways with metabolism. Chapters 11 and 12 from Guillaume et al. and
Püschel and Munoz-Pinedo identify crosstalk between nutrient metabolism with autophagy
and cell death pathways, respectively. Often, cellular functionality is directly linked to
v
vi Preface
metabolism. In Chapters 13 and 14, Liu and Ho and Fernandez Garcia and Fendt provide
protocols to assess the regulation exerted by nutrient availability on macrophage polariza-
tion and T-lymphocyte metabolism, respectively.
To further understand and exploit metabolism in systems medicine, different model
systems and cellular contexts can be considered. An important model system used in cancer
research are patient-derived xenografts. Annibali and colleagues describe in Chapter 15 the
setup of patient-derived xenografts, which can be used to determine in vivo tumor metabo-
lism as described in Chapter 5. Moreover, it has been established that tumor metabolism
changes during cancer progression. Therefore, it is important to closely follow cancer
progression, as described in Chapter 16 by Stanchi and colleagues using microscopy.
Further, external signals and stimuli are in crosstalk with metabolism. An important con-
tributor hereby is visceral adipose tissue (VAT), an active endocrine organ producing
hormones. To study the impact of VAT-produced hormones on metabolism, VAT can be
surgically removed. A protocol to do so is described in Chapter 17 by Chakraborty and
Bernard. Similarly, the secretome of other organs such as the bone is a function of health and
disease. Potential interaction of this secretome with metabolism therefore requires quanti-
tative methods to define bone status and its secretome. A method hereof is described by Lie
and colleagues in Chapter 18. Moving from a whole-body physiology level to the cellular
level, the relevance of organelle organization emerges, and it is tempting to speculate that
there might be a crosstalk between organelle organization and metabolic fluxes within
organelles (see Chapter 10). A protocol provided by Latge and Schauer in Chapter 19 allows
determination of intracellular organelle organization. Finally, large-scale data often obtained
when studying metabolism can be effectively displayed using heat maps. Fundamentals of
constructing and interpreting heat maps are provided by Vacanti in Chapter 20.
With this book, we aim to provide researchers with methods to study, perturb and
functionally interpret metabolic signaling from the subcellular to the whole-body level.
Given the emerging importance of metabolism in sustaining health and metabolic deregu-
lation in disease, we believe that applying methods described in this book will foster
mechanistic understanding of metabolism and, in the long-term, prospectively support
the development of innovative disease treatment strategies.
Leuven, Belgium Sarah-Maria Fendt

East Lansing, MI, USA Sophia Y. Lunt
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 A Protocol to Compare Methods for Untargeted Metabolomics . . . . . . . . . . . . . . 1
Lingjue Wang, Fuad J. Naser, Jonathan L. Spalding, and Gary J. Patti
2 High-Throughput Measure of Bioactive Lipids Using Non-targeted
Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Kim A. Lagerborg, Jeramie D. Watrous, Susan Cheng, and Mohit Jain
3 Measuring the Nutrient Metabolism of Adherent Cells in Culture . . . . . . . . . . . . 37
Martin P. Ogrodzinski, Shao Thing Teoh, Lei Yu, Deanna Broadwater,
Elliot Ensink, and Sophia Y. Lunt
13
4 C Tracer Analysis and Metabolomics in 3D Cultured Cancer Cells . . . . . . . . . . 53
Marit van Gorsel, Ilaria Elia, and Sarah-Maria Fendt
5 Measuring In Vivo Tissue Metabolism Using 13C Glucose Infusions
in Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Dorien Broekaert and Sarah-Maria Fendt
6 Measuring Human Lipid Metabolism Using Deuterium Labeling:
In Vivo and In Vitro Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Katherine E. Pinnick, Pippa J. Gunn, and Leanne Hodson
7 Measuring Rates of ATP Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Matthew J. Bird, Silvia Radenkovic, Pieter Vermeersch,
and David Cassiman
8 Direct Estimation of Metabolic Flux by Heavy Isotope Labeling
Simultaneous with Pathway Inhibition: Metabolic Flux Inhibition Assay . . . . . . . 109
Tong Zhang, Christiaan F. Labuschagne, Karen H. Vousden,
and Oliver D. K. Maddocks
9 Measuring Glycolytic and Mitochondrial Fluxes in Endothelial Cells
Using Radioactive Tracers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Koen Veys, Abdiel Alvarado-Diaz, and Katrien De Bock
10 Determining Compartment-Specific Metabolic Fluxes. . . . . . . . . . . . . . . . . . . . . . . 137
Yannic Nonnenmacher, Roberta Palorini, and Karsten Hiller
11 Determining the Impact of Metabolic Nutrients on Autophagy . . . . . . . . . . . . . . 151
Jessica D. Guillaume, Stephanie L. Celano, Katie R. Martin,
and Jeffrey P. MacKeigan
12 Measuring the Activation of Cell Death Pathways upon Inhibition
of Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Franziska Püschel and Cristina Muñoz-Pinedo
13 Determining Macrophage Polarization upon Metabolic Perturbation . . . . . . . . . 173
Pu-Ste Liu and Ping-Chih Ho
vii
viii Contents
14 Assessing the Impact of the Nutrient Microenvironment

on the Metabolism of Effector CD8+ T Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Juan Fernández Garcı́a and Sarah-Maria Fendt
15 Development of Patient-Derived Tumor Xenograft Models . . . . . . . . . . . . . . . . . . 217
Daniela Annibali, Eleonora Leucci, Els Hermans,
and Frédéric Amant
16 Imaging Glioma Progression by Intravital Microscopy. . . . . . . . . . . . . . . . . . . . . . . 227
Fabio Stanchi, Ken Matsumoto, and Holger Gerhardt
17 Lipectomizing Mice for Applications in Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . 245
Debrup Chakraborty and Jamie J. Bernard
18 Quantitative Multiplex Immunoassay for Profiling Bone
Turnover Biomarkers in Human Bone Tissue Culture Supernatants . . . . . . . . . . . 251
Wen-Rong Lie, Derek F. Amanatullah, and Bonnie L. King
19 Determining the Intracellular Organization of Organelles . . . . . . . . . . . . . . . . . . . 263
Bruno Latgé and Kristine Schauer
20 The Fundamentals of Constructing and Interpreting Heat Maps . . . . . . . . . . . . . 279
Nathaniel M. Vacanti
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
Contributors
ABDIEL ALVARADO-DIAZ Laboratory of Exercise and Health, Department of Health Sciences

and Technology, ETH Zürich, Zürich, Switzerland
DEREK F. AMANATULLAH Department of Orthopaedic Surgery, Stanford University School of
Medicine, Redwood City, CA, USA
FRÉDÉRIC AMANT Gynecological Oncology, Oncology Department, LKI Leuven Cancer
Institute KU Leuven-University of Leuven, Leuven, Belgium; Centre for Gynecologic
Oncology Amsterdam (CGOA), Antoni Van Leeuwenhoek-Netherlads Cancer Institute
(AvL-NKI) and University Medical Centra (UMC), Amsterdam, The Netherlands
DANIELA ANNIBALI Gynecological Oncology, Oncology Department, LKI Leuven Cancer
Institute KU Leuven-University of Leuven, Leuven, Belgium
JAMIE J. BERNARD Department of Pharmacology and Toxicology, Michigan State University,
East Lansing, MI, USA
MATTHEW J. BIRD Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium;
Hepatology Laboratory, Department of Chronic Diseases, Metabolism and Ageing,
KU Leuven, Leuven, Belgium
DEANNA BROADWATER Department of Biochemistry and Molecular Biology, Michigan State
University, East Lansing, MI, USA
DORIEN BROEKAERT Laboratory of Cellular Metabolism and Metabolic Regulation,
VIB Center for Cancer Biology, VIB, Leuven, Belgium; Laboratory of Cellular Metabolism
and Metabolic Regulation, Department of Oncology, Leuven Cancer Institute (LKI),
DAVID CASSIMAN Hepatology Laboratory, Department of Chronic Diseases, Metabolism and
Ageing, KU Leuven, Leuven, Belgium; Metabolic Center, University of Leuven, Leuven,
Belgium
STEPHANIE L. CELANO College of Human Medicine, Michigan State University, Grand
Rapids, MI, USA
DEBRUP CHAKRABORTY Department of Pharmacology and Toxicology, Michigan State
SUSAN CHENG Cardiovascular Division, Department of Medicine, Brigham and Women’s
Hospital, Harvard Medical School, Boston, MA, USA; Framingham Heart Study,
Framingham, MA, USA
KATRIEN DE BOCK Laboratory of Exercise and Health, Department of Health Sciences and
Technology, ETH Zürich, Zürich, Switzerland
ILARIA ELIA Laboratory of Cellular Metabolism and Metabolic Regulation, VIB Center for
Cancer Biology, VIB, Leuven, Belgium; Laboratory of Cellular Metabolism and Metabolic
Regulation, Department of Oncology, Leuven Cancer Institute (LKI), KU Leuven,
Leuven, Belgium
ELLIOT ENSINK Department of Biochemistry and Molecular Biology, Michigan State
SARAH-MARIA FENDT Laboratory of Cellular Metabolism and Metabolic Regulation,
Department of Oncology, VIB-KU Leuven Center for Cancer Biology, Leuven, Belgium
ix
x Contributors
JUAN FERNÁNDEZ-GARCÍA Laboratory of Cellular Metabolism and Metabolic Regulation,

VIB Center for Cancer Biology, VIB, Leuven, Belgium; Laboratory of Cellular Metabolism
HOLGER GERHARDT VIB-KU Leuven Center for Cancer Biology (CCB), Leuven, Belgium;
Max Delbrück Center for Molecular Medicine, Berlin, Germany
JESSICA D. GUILLAUME College of Human Medicine, Michigan State University, Grand
Rapids, MI, USA
PIPPA J. GUNN Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill
Hospital, University of Oxford, Oxford, UK
ELS HERMANS Trace, Department of Oncology, LKI Leuven Cancer Institute KU Leuven-
University of Leuven, Leuven, Belgium; VIB-KU Leuven Center for Cancer Biology,
Leuven, Belgium
KARSTEN HILLER Department of Bioinformatics and Biochemistry, Braunschweig
Integrated Center of Systems Biology (BRICS), Technische Universit€at Braunschweig,
Braunschweig, Germany; Computational Biology of Infection Research, Helmholtz Centre
for Infection Research, Braunschweig, Germany
PING-CHIH HO Department of Fundamental Oncology, Faculty of Biology and Medicine,
University of Lausanne, Epalinges, Vaud, Switzerland; Ludwig Lausanne Branch,
Epalinges, Vaud, Switzerland
LEANNE HODSON Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill
Hospital, University of Oxford, Oxford, UK
MOHIT JAIN Department of Medicine, University of California, San Diego, La Jolla, CA,
USA; Department of Pharmacology, University of California, San Diego, La Jolla, CA,
USA
BONNIE L. KING Department of Pediatrics, Stanford University School of Medicine,
Stanford, CA, USA
CHRISTIAAN F. LABUSCHAGNE The Francis Crick Institute, London, UK
KIM A. LAGERBORG Department of Medicine, University of California, San Diego, La Jolla,
CA, USA; Department of Pharmacology, University of California, San Diego, La Jolla,
CA, USA
BRUNO LATGÉ Molecular Mechanisms of Intracellular Transport Group, Institut Curie,
PSL Research University, Paris Cedex 05, France; Centre National de la Recherche
Scientifique, Unité Mixte de Recherche 144, Paris, France
ELEONORA LEUCCI Trace, Department of Oncology, LKI Leuven Cancer Institute
KU Leuven-University of Leuven, Leuven, Belgium; Laboratory of RNA Cancer Biology,
Department of Oncology, LKI Leuven Cancer Institute KU Leuven-University of Leuven,
Leuven, Belgium
WEN-RONG LIE MilliporeSigma Corporation, St Louis, MO, USA
PU-STE LIU Department of Fundamental Oncology, Faculty of Biology and Medicine,
University of Lausanne, Epalinges, Vaud, Switzerland; Ludwig Lausanne Branch,
Epalinges, Vaud, Switzerland; Institute of Cellular and System Medicine, National Health
Research Institutes, Miaoli County, Taiwan
SOPHIA Y. LUNT Department of Biochemistry and Molecular Biology, Michigan State
University, East Lansing, MI, USA; Department of Chemical Engineering and Materials
Science, Michigan State University, East Lansing, MI, USA
JEFFREY P. MACKEIGAN College of Human Medicine, Michigan State University, Grand
Rapids, MI, USA
Contributors xi
OLIVER D. K. MADDOCKS Wolfson Wohl Cancer Research Centre, Institute of Cancer

Sciences, University of Glasgow, Glasgow, UK
KATIE R. MARTIN College of Human Medicine, Michigan State University, Grand Rapids,
MI, USA
KEN MATSUMOTO VIB-KU Leuven Center for Cancer Biology (CCB), Leuven, Belgium
CRISTINA MUÑOZ-PINEDO Oncobell Program, Cell Death Regulation Group, Bellvitge
Biomedical Research Institute (IDIBELL), Barcelona, Spain
FUAD J. NASER Department of Chemistry, Washington University, St. Louis, MO, USA
YANNIC NONNENMACHER Department of Bioinformatics and Biochemistry, Braunschweig
Integrated Center of Systems Biology (BRICS), Technische Universit€
at Braunschweig,
Braunschweig, Germany
MARTIN P. OGRODZINSKI Department of Biochemistry and Molecular Biology, Michigan
State University, East Lansing, MI, USA; Department of Physiology, Michigan State
ROBERTA PALORINI Department of Biotechnology and Biosciences, University of Milano-
Bicocca, Milan, Italy
GARY J. PATTI Department of Chemistry, Washington University, St. Louis, MO, USA;
Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA
KATHERINE E. PINNICK Oxford Centre for Diabetes, Endocrinology and Metabolism,
Churchill Hospital, University of Oxford, Oxford, UK
FRANZISKA PÜSCHEL Oncobell Program, Cell Death Regulation Group, Bellvitge Biomedical
Research Institute (IDIBELL), Barcelona, Spain
SILVIA RADENKOVIC Hepatology Laboratory, Department of Chronic Diseases, Metabolism
and Ageing, KU Leuven, Leuven, Belgium; Metabolomics Expertise Center, VIB-KU
Leuven Center for Cancer Biology, Leuven, Belgium
KRISTINE SCHAUER Molecular Mechanisms of Intracellular Transport Group, Institut
Curie, PSL Research University, Paris Cedex 05, France; Centre National de la Recherche
Scientifique, Unité Mixte de Recherche 144, Paris, France
JONATHAN L. SPALDING Department of Chemistry, Washington University, St. Louis, MO,
USA; Department of Medicine, Washington University School of Medicine, St. Louis, MO,
USA
FABIO STANCHI VIB-KU Leuven Center for Cancer Biology (CCB), Leuven, Belgium
SHAO THING TEOH Department of Biochemistry and Molecular Biology, Michigan State
NATHANIEL M. VACANTI Division of Nutritional Sciences, Cornell University, Ithaca, NY,
USA; Department of Oncology-Pathology, Science for Life Laboratory, Karolinska
Institutet, Stockholm, Sweden
MARIT VAN GORSEL Laboratory of Cellular Metabolism and Metabolic Regulation, VIB
Center for Cancer Biology, VIB, Leuven, Belgium; Laboratory of Cellular Metabolism
PIETER VERMEERSCH Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium
KOEN VEYS Laboratory of Angiogenesis and Vascular Metabolism, VIB Center for Cancer
Biology, VIB, Leuven, Belgium; Laboratory of Angiogenesis and Vascular Metabolism,
Department of Oncology, KU Leuven, Leuven, Belgium
KAREN H. VOUSDEN The Francis Crick Institute, London, UK
LINGJUE WANG Department of Chemistry, Washington University, St. Louis, MO, USA
xii Contributors
JERAMIE D. WATROUS Department of Medicine, University of California, San Diego,

La Jolla, CA, USA; Department of Pharmacology, University of California, San Diego,
La Jolla, CA, USA
LEI YU Department of Biochemistry and Molecular Biology, Michigan State University,
East Lansing, MI, USA
TONG ZHANG Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences,
University of Glasgow, Glasgow, UK
Chapter 1
A Protocol to Compare Methods for Untargeted

Metabolomics
Lingjue Wang, Fuad J. Naser, Jonathan L. Spalding, and Gary J. Patti
Abstract
There are thousands of published methods for profiling metabolites with liquid chromatography/mass
spectrometry (LC/MS). While many have been evaluated and optimized for a small number of select
metabolites, very few have been assessed on the basis of global metabolite coverage. Thus, when performing
untargeted metabolomics, researchers often question which combination of extraction techniques, chro-
matographic separations, and mass spectrometers is best for global profiling. Method comparisons are
complicated because thousands of LC/MS signals (so-called features) in a typical untargeted metabolomic
experiment cannot be readily identified with current resources. It is therefore challenging to distinguish
methods that increase signal number due to improved metabolite coverage from methods that increase
signal number due to contamination and artifacts. Here, we present the credentialing protocol to remove
the latter from untargeted metabolomic datasets without having to identify metabolite structures. This
protocol can be used to compare or optimize methods pertaining to any step of the untargeted metabo-
lomic workflow (e.g., extraction, chromatography, mass spectrometer, informatic software, etc.).
Key words Untargeted metabolomics, Metabolite profiling, Metabolism, Credentialing, Liquid

chromatography, Mass spectrometry
1 Introduction
Metabolite profiling, or metabolomics, can be performed with

either a targeted or an untargeted approach. In targeted metabo-
lomics, a defined set of metabolites is analyzed. As such, these
methods are relatively straightforward to optimize by using com-
mercial standards [1]. In contrast to targeted metabolomics, which
is effective at testing specific hypotheses, the objective of untar-
geted metabolomics is to measure as many metabolites in the
sample as possible [2]. This systems-level assessment of metabolism
is highly attractive because it can potentially reveal altered pathways
that had not been previously anticipated [3]. Since the set of
metabolites being profiled is not well defined and may include
“unknown” compounds that have not yet been characterized,
Sarah-Maria Fendt and Sophia Y. Lunt (eds.), Metabolic Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1862,
https://doi.org/10.1007/978-1-4939-8769-6_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019
1
2 Lingjue Wang et al.
however, simple optimization of untargeted metabolomic methods

with commercial standards is impractical.
In principle, untargeted metabolomic methods can be opti-
mized by maximizing the number of metabolites detected in an
experiment. In practice, this approach is complicated because it is
typical to detect thousands of signals (or features) in an untargeted
metabolomic experiment that cannot be identified with current
informatic workflows [4]. While some of these unidentified signals
arise from bona fide metabolites in the biological sample, others do
not and therefore the total number of detected signals does not
reliably correlate with metabolome coverage [5]. Non-biological
signals arise due to contamination and artifacts. Contaminants are
chemical impurities introduced during sample handling and
LC/MS analysis (e.g., solvent impurities, plastic leechables in the
extraction process, carry over from previous experiments, etc.).
Artifacts are signals that result from informatic errors. In some
untargeted metabolomic experiments, contaminants and artifacts
may represent a major fraction of the total LC/MS signals
detected [6].
Considering the number of extraction protocols, separation
methods, mass spectrometers, and informatic software packages,
there are thousands of potential workflows for performing untar-
geted metabolomics [7–11]. A fundamental question is which
combination of techniques, instrumentation, and equipment set-
tings is best for achieving comprehensive metabolome coverage.
New laboratories trying to establish an untargeted metabolomic
platform, for example, may wish to compare the performance of
different mass spectrometers prior to committing financial
resources.
Historically, metabolite coverage in untargeted metabolomics
has often been benchmarked by the total number of signals
detected. It is important to emphasize that such an approach is
highly unreliable because the total number of signals and the total
number of metabolites poorly correlate [5]. By way of illustration, a
method using dirty vials may increase the number of signals (as a
result of contamination) without increasing the actual number of
metabolites assayed. As an alternative approach to better assess
metabolome coverage in LC/MS experiments without having to
identify each signal, we present the credentialing protocol using
E. coli as a representative complex biological sample. In brief,
credentialing mixes uniformly labeled E. coli extracts with natural-
abundance E. coli extracts at different ratios. Signals in the LC/MS
data that correspond to bona fide metabolites will have an isotopic
dance partner at the appropriate ratio, whereas signals
corresponding to contaminants and artifacts will not. After remov-
ing contaminants and artifacts, the number of remaining signals can
then be used as a better estimate of metabolite coverage between
different untargeted metabolomic methods [12].
Optimizing Methods for Untargeted Metabolomics 3
The following protocol consists of two main parts. First, we

describe how to make credentialed samples (this part can be skipped
when using a purchased credentialing kit). Then, we describe in
detail how to process credentialing data. The output of this work-
flow will be a list of signals or features from which non-credentialed
contaminants and artifacts have been removed. We note that the
approach we present for data analysis can be applied to any set of
appropriately labeled biological materials (e.g., plants, mammalian
cells, animal tissues, etc.). Given the costs and experimental chal-
lenges of generating such samples, however, we focus on using
E. coli here as a representative complex biological matrix for
method optimization. E. coli samples that have been labeled for
credentialing analysis can be commercially obtained from Cam-
bridge Isotope Laboratories.
2 Materials
Prepare all solutions and perform cell culture with ultrapure water.
All glassware referred to in this protocol should be sterile unless
noted otherwise. Sterilization can be achieved prior to E. coli
growth by thoroughly rinsing glassware with ultrapure water, cov-
ering with aluminum foil, and baking in an oven at 250 C for
3–5 h.
2.1 Purchasing Metabolic extracts of credentialed E. coli (strain K12, MG1655)

Metabolic Extracts may be purchased from Cambridge Isotope Laboratories (Tewks-
of Credentialed E. coli bury, MA, USA). Cambridge offers two Credentialed E. coli Cell
Extract products: an extract kit in solution (Item #: MSK-CRED-
KIT) and a dried down extract kit (Item #: MSK-CRED-DD-KIT).
Both kits contain separate vials of unlabeled extract and uniformly
13
C-labeled extract. Products in solution have been suspended in
acetonitrile:water (1:1). E. coli have been extracted as previously
detailed [13].
2.2 Media 1. 5 M9 minimal salts. Final concentration: 33 g/L disodium

Preparation phosphate, 15 g/L monopotassium phosphate, 2.5 g/L
for Credentialed E. coli sodium chloride, and 5 g/L ammonium chloride.
Growth 2. Lennox B broth powder (10 g/L enzymatic digest of casein,
5 g/L yeast extract, and 5 g/L sodium chloride).
3. 1 M magnesium sulfate.
4. 0.1 M calcium chloride.
5. Ultrapure water.
6. Two 500 mL Erlenmeyer flasks.
7. 500 mL media filter with a 0.22 μm pore size.
8. 250 mL media filter with a 0.22 μm pore size.

9. Parafilm.
10. Vacuum suction pump.
2.3 Initial E. coli 1. E. coli stock strain K12, MG1655. Store at 80 C and do not
Overnight Growth let the stock thaw.
2. Styrofoam box.
3. Ice.
4. Two 250 mL Erlenmeyer flasks.
5. 50 mL LB media.
6. Sterile Pasteur pipets.
7. Rotary shaker.
2.4 Credentialed 1. Two 10 mL beakers.

E. coli Growth 2. Natural abundance D-glucose (200 mg).
3. Uniformly labeled 13C-D-glucose (207 mg).
4. Ultrapure water.
5. Charged auto-pipetter.
6. Two 25 mL disposal plastic pipettes.
7. M9 media (220 mL).
8. Two 0.22 μm syringe filters.
9. Two 3 mL plastic syringes.
10. Two 1 L Erlenmeyer flasks.
11. Rotary shaker.
12. Pipette and pipette tips (1 mL).
13. UV-Vis spectrophotometer.
14. Spectrophotometer cuvettes.
2.5 Credentialed 1. Two 50 mL conical tubes.

E. coli Harvest 2. Centrifuge for 50 mL conical tubes.
3. Charged auto-pipetter.
4. Disposable plastic pipettes (25 mL or 50 mL).
5. Liquid nitrogen.
6. Ice.
7. Lyophilizer.
8. Eppendorf tubes.
2.6 LC/MS 1. LC/MS-grade solvents (e.g., water, acetonitrile, methanol,

and isopropanol). We recommend solvents from Honeywell
Burdick & Jackson (Muskegon, MI, USA).
2. LC/MS-grade eluent additives such as ammonium acetate. We
recommend purchasing mobile phase additives from Sigma
Aldrich (St. Louis, MO, USA).
3. LC/MS method of your choice.
2.7 Data Processing 1. R and RStudio. See Subheading 4.1 for more details about R
and Analysis installation.
2. The following R packages: Credential3.1, data.table, utils, and
xcms (or an equivalent software package for generating a fea-
tures table).
3. ProteoWizard MSConvert (if using the XCMS workflow).
3 Methods
3.1 E. coli Media 1. Prepare M9 media and LB media 1 day before E. coli harvest.
Preparation 2. Combine 500 mL of ultrapure water and 5.5 g of 5 M9
minimal salts in a sterile 500 mL Erlenmeyer flask.
3. Cover with parafilm and mix by inversion. Ensure that salts are
fully dissolved before filtration step (about 5 min).
4. Add via pipette 500 μL of 1 M MgSO4 and 250 μL of 0.1 M
CaCl2.
5. Cover with parafilm and mix by inversion.
6. Filter via vacuum suction through a 500 mL media filter bottle.
7. In a separate 500 mL Erlenmeyer flask, combine 250 mL ultra-
pure water and 5 g of LB powder.
8. Cover with parafilm and mix by inversion. Ensure that powder
is fully dissolved before filtration step (about 5 min).
9. Filter via vacuum suction through a 250 mL media filter.
3.2 Initial E. coli 1. We recommend performing this portion of the experiment by

Overnight Growth 5:00 pm the day before harvest to give adequate time for initial
overnight growth and subsequent credentialed E. coli cultures.
2. Remove E. coli stock from 80 C, making sure not to let the
stock thaw. Place it directly on ice in a Styrofoam box.
3. Label one 250 mL Erlenmeyer flask as “control” and one
as “E. coli.”
4. Add 25 mL of LB media to each flask by pouring.
5. Swirl a Pasteur pipette in the “control” flask.
6. Inoculate the “E. coli” flask with E. coli stock by pressing a
sterile Pasteur pipette into the frozen stock. Verify that some of
the stock slush is attached to the pipette tip. Swirl the pipette
tip in the “E. coli” flask, using a twisting of the finger at the top
of the Pasteur pipette to evacuate the pipette of any stock
liquid.
7. Place both flasks into the rotary shaker overnight at 300 rpm
and 37 C.
8. From this point forward, all manipulations are quantitative.
Great care should be taken to ensure each of the credentialed
E. coli cultures are treated identically.
3.3 Glucose Solution 1. Verify that the control overnight culture has not been
and E. coli Culture contaminated.
Preparation 2. The morning following overnight inoculation, label two
25 mL Erlenmeyer flasks as “12C glucose” and “13C glucose.”
3. Add 2 mL of ultrapure water to each of the 25 mL flasks.
4. To the “12C glucose” flask, add 200 mg of natural abundance
D-glucose.
5. To the “13C glucose” flask, add 207 mg of uniformly labeled

13
C-D-glucose.
6. Cover both flasks with parafilm.
7. Swirl gently and let it sit at room temperature until solutes are
fully dissolved in water. This step may take up to 10 min. Do
not let the glucose solutions come into contact with anything.
Do not let any solution spill out of the flasks.
8. Label one sterile 1 L Erlenmeyer flask as “12C” and another as
“13C.”
9. Via auto-pipetter and a disposable 25 mL pipette, add 100 mL
of M9 media to each flask.
10. Via syringe, remove all of the natural abundance glucose solu-
tion, filling the remainder of the syringe with air.
11. Attach a syringe filter to the syringe.
12. Slowly dispense the solution into the “12C” flask while holding
the filter in place. Verify final dispensing is air to ensure com-
plete transfer of glucose.
13. Add 1 mL of ultrapure water to the empty 25 mL flask and
swirl.
14. Remove the syringe filter and place it in its package. As above,
remove the 1 mL of water and dispense through the same filter
to ensure complete transfer.
13
15. Repeat steps 10–14 for the uniformly labeled C-D-glucose
solution, using a fresh syringe and filter.
3.4 Credentialed 1. Take the OD600 of the overnight culture by adding approxi-
E. coli Growth mately 0.5 mL to a clear, plastic cuvette and taking an absor-
bance reading with a UV-VIS spectrophotometer. Be sure to
blank the absorbance reading with leftover M9 media.
2. If the absorbance is greater than 1.0, dilute a small volume of
the overnight culture 10 in water until the OD600 is below
1.0. Calculate OD600Effective by multiplying the final absor-
bance reading by the dilution factor.
3. Calculate the desired volume of overnight culture to be added
to each 1 L flask by the following equation: 1.5 mL/
(OD600Effective/1000) ¼ μL of overnight culture to add.
The volume should be less than 1 mL but greater than 50 μL. If
less than 100 μL is calculated, continue as written, but realize
that growth times may be extended.
4. Add the calculated volume of overnight culture to each of the
1 L flasks via pipette. This step is critical to ensuring your cultures
grow at the same rate. Any error here is multiplied exponentially.
Be sure to swirl the overnight culture before pipetting. Pipette and
dispense twice into the overnight culture before pipetting into the
1 L culture flasks. Pipette directly into the culture, not down the
side of the flask. Be mindful of extraneous drops.
5. Place both flasks into the rotary shaker at 300 rpm and 37 C.
6. Monitor growth with OD600 measurements until
OD600 ¼ 0.7, which is ready for harvest. Cultures should
remain within 0.05 OD600 of each other at harvest time. If
necessary, harvest at different times such that OD600 values are
equal.
(a) Blank the UV-VIS spectrophotometer with leftover M9
media.
(b) We suggest taking readings every 2 h until OD600 ¼ 0.5,
every 1 h until OD600 ¼ 0.6, and every 15 min until
OD600 ¼ 0.7.
3.5 Credentialed 1. Minimize the time between removal of the cultures from incu-
E. coli Harvest bation and freezing of the pellets in liquid nitrogen. Keep this
time constant between batches.
2. Place 50 mL conical tubes on ice in preparation for harvest.
3. Remove the natural abundance and 13C-labeled E. coli culture
flasks from rotary shaker and place on ice.
4. Swirl and pipette 50 mL of the cultures into each tube using
separate 50 mL disposable pipettes, noting the actual volume in
the final tube.
5. Centrifuge at 0 C and 3200 rcf for 10 min.
6. Decant the supernatant, taking care not to disturb the pellets.
7. Rinse the top of the pellet with 0.5 mL of ultrapure water by

pipetting gently down the side of the tube at an incline. Decant
the supernatant.
8. Place conical tubes upright in liquid nitrogen, completely
freezing the pellets.
9. Transfer conical tubes to the lyophilizer, taking care to main-
tain liquid nitrogen temperatures. Remove lids and cover the
tops of each tube with Kimwipes, secured with rubber bands.
10. Dry on the lyophilizer for 24 h, or until completely dry.
11. Weigh powder into separate 1.5 mL Eppendorf tubes and
transfer to a 80 C freezer.
3.6 Credentialed 1. Perform the metabolite extraction protocol of choice (for

E. coli Extraction credentialed E. coli growth samples only). The amount of dry
and LC/MS mass extracted may vary depending on the protocol, signal-to-
noise of the mass spectrometer being used, number of
replicates, etc.
2. Mix the E. coli extracts to create two credentialed samples. The
first sample should have natural-abundance E. coli and uni-
formly labelled E. coli in a 1:1 ratio by volume. The second
sample should have natural-abundance E. coli and uniformly
labeled E. coli in a 1:2 ratio by volume.
3. To concentrate the credentialed samples, dry and reconstitute
the credentialed extracts in solution by using your method of
choice.
If E. coli standard extracts were purchased in solution from
Cambridge Isotopes, mix the extracts in ratios as detailed
in step 2. If E. coli standard extracts were purchased from
Cambridge Isotopes as a powder, reconstitute the extracts as
indicated in step 3 and mix the reconstituted extracts in the
ratios as indicated in step 2.
4. Transfer reconstituted extracts to LC/MS vials.
5. Perform LC/MS using your choice of chromatography, mass
spectrometer, data acquisition settings, etc.
3.7 LC/MS Data Credentialing is compatible with most data-processing software, as

Analysis illustrated in Fig. 1. In this protocol, we detail a method using
XCMS for feature detection, since it is commonly used in untar-
geted metabolomics [14]. We also include details on how to use
other data-processing pipelines with credentialing.
1. Data processing will likely take less than 2 h from this point
forward (depending on computer-processing speed).
2. Data analysis will be performed with RStudio. See Subheading
4.1 to install R and R packages on your computer.
raw LC/MS data Stage 1: LC/MS Data Collection

(.d,.RAW, .WIFF, etc)
ProteoWizard MSconvert
Stage 2: Feature Detection
.mzXML files other data processing pipelines
XCMS
Stage 3: Credentialing
feature tables
in data.table format
credentialing
credentialed features
Fig. 1 Three stages of the credentialing workflow
3. A detailed template is available online at the link below to

assist you in data processing with XCMS and credentialing:
https://github.com/pattilabwu/Credential3.1/blob/master/Sam
pleScript.R.
3.7.1 Loading R 1. In the RStudio software, run the following to load the required
Packages and Setting Up R packages:
a Working Directory
library(xcms)
library(Credential3.1)
library(data.table)
library(utils)
2. Use one of the following to set up a working directory, depend-

ing on your operating system:
setwd("/Users/Lingjue/CredentialingDemo") # MacOS format
setwd("C:/Users/Mike/Desktop/CredentialingDemo") # Windows format
3. Note that every time a new R session is opened, all required

packages should be loaded again.
4. If you are using a data-processing pipeline that does not rely

upon XCMS for peak detection, you may skip Subheading
3.7.2.
3.7.2 Data Processing 1. Open MSConvert.

with XCMS 2. On the file loading panel, click “Browse” to select all data files.
MS Raw Data Conversion 3. Select output directory.
4. In the “Filters” panel, select “Peak Picking” and click “Add” to
add the filter.
5. Remove all default filters, such as “titleMaker.”
6. Click “Start” to begin converting raw data into mzXML
format.
7. Figure 2 shows the Graphical User Interface of MSConvert and
highlights the relevant entries related to each step above.
Feature Detection 1. Sort mzXML files for the credentialed samples into two folders
based on natural abundance and uniformly labeled mixing
ratios (e.g., folders are labeled as “1T1” for 1:1 mixing
ratio and “1T2” for 1:2 mixing ratio). The folders should be
in the working directory you set up in Subheading 3.7.1.
XCMS processing includes peak detection, feature grouping,
Fig. 2 Screenshot illustrating the conversion of raw MS data with MSConvert

retention time correction, and missing peak filling. Run the

following to process the mzXML files in the 1:1 mixing ratio
folder (“1T1”):
# 1. peak detection with centWave algorithm
xs_1 = xcmsSet("./1T1", method="centWave", ppm=20, peak-
width = c(10,30), snthresh=5, prefilter=c(3,100))
# 2. initial peak grouping, 1st round
xs_1= group(xs_1, bw=5, mzwid=.015, minfrac=0.5)
# 3. retention time correction
xs_1 = retcor(xs_1, method="obiwarp",profStep=1)
# 4. feature grouping, 2nd round
xs_1 = group(xs_1, bw=5, mzwid=.015, minfrac=0.5)
# 5. filling missing peaks
xs_1 = fillPeaks(xs_1)
2. Repeat step 1 for the 1:2 mixing ratio files (in “1T2” folder).
That is, copy and paste the above code and change xs_1 to xs_2
(or any other name) and “./1T1” to “./1T2”.
Please note that users need to adjust XCMS parameters based
on their instrumentation and methods. For further information,
see Subheading 4.2 below. For detailed documentation about XCMS
parameters, see: https://bioconductor.org/packages/release/bioc/
manuals/xcms/man/xcms.pdf.
3.7.3 Input Format The format of input data for credentialing is a data.table object
for Credentialing with only four columns: “cc”—feature index number, “rt”—reten-
tion time, “mz”—m/z ratio, and “i”—signal intensity. The steps
below describe how to create such an object.
Automated Processing xsGroupExtract() is a function in the Credential3.1 R package. It

of XCMS Files by extracts features from the xcmsSet object and adjusts the format for
Credentialing credentialing. Run the following to extract features in xs_1 and xs_2
from Subheading 3.7.2 “Feature Detection”:
featureGroup1 = xsGroupExtract(xs_1,intchoice = "into", sampling = 1)
featureGroup2 = xsGroupExtract(xs_2,intchoice = "into", sampling = 1)
features1T1 = data.table(featureGroup1$credTable)
features1T2 = data.table(featureGroup2$credTable)
*Use the help(xsGroupExtract) command to see the description of

parameters and output of xsGroupExtract().
Manual Processing of Files If data processing was performed by another software platform:
from Other Data Processing *Important: when using other data processing pipelines, do
Pipelines not apply any function to group or remove isotope patterns.
1. Export the resulting features from each mixing ratio condition
(“1T1” and “1T2”) to two CSV tables. The tables should
Fig. 3 Screenshot of a CSV file with appropriate format for credentialing
include only four columns: “cc”, “mz”, “rt,” and “i”. An

example is shown in Fig. 3.
2. Place the CSV files in the working directory and import them
with the following:
features1T1 = data.table(read.csv(“features1T1.csv”))
features1T2 = data.table(read.csv(“features1T2.csv”))
3.7.4 Credentialing Run the following to perform credentialing:
credential = credentialing(features1T1,features1T2,ppm = 20,

rtwin = 2,rtcom = 5, ratio1 = 1/1, ratio2 = 1/2, ratio_tol =
0.1, ratios_tol = 0.2)
*Parameters of the credentialing() function:

l ppm—mass error tolerance for isotope pair searching and
grouping.
l rtwin—retention time window in the first round of credentialed
feature selection.
l rtcom—retention time window in the second round of
credentialed feature selection.
l ratio1—mixing ratio of unlabeled to labeled extract for the first
credentialed sample (Default: 1/1).
l ratio2—mixing ratio of unlabeled to labeled extract for the second
credentialed sample (Default: 1/2).
Table 1
Select tables generated by the credentialing() function
Name Content
CredentialedFeatureR2F Final credentialed features with second ratio filter
CredentialedFeatureR2 Final credentialed features without second ratio filter
CredentialedFeature1N2 First credentialed features from “1T1” excluded by second credentialing
CredentialedFeature2N2 First credentialed features in “1T2” excluded by second credentialing
CredentialedFeature1R1 First credentialed features from “1T2” (1/1 ratio)
CredentialedFeature2R1 First credentialed features from “1T2” (1/2 ratio)
l ratio_tol—a factor between [0, 1] that sets the acceptable range for
the intensity ratio relative to the actual mixing ratio (Default:
0.1).
l ratios_tol—a factor between [0, 1] that sets the acceptable range
for the intensity ratio relative to the combined mixing ratio
(ratio1/ratio2, Default: 0.2).
*Generally, users should set these parameters based on their
LC/MS instrumentation and methods. For more information
regarding parameters, see: https://github.com/pattilabwu/Creden
tial3.1/blob/master/README.md.
3.7.5 Data Output The output of the credentialing function in R is a list object that
and Interpretation includes credentialed and non-credentialed features. By typing in
credential$ and hitting the “tab” button, a list of tables will
appear. Press the enter key to display the highlighted table. Some
of the tables are listed in Table 1.
For more information regarding the output of credentialing,
see: https://github.com/pattilabwu/Credential3.1/blob/mas-
ter/README.md.
4 Notes
4.1 R Initiation, 1. To install R, see: https://cran.r-project.org/.

Package Installation, 2. RStudio is the recommended working environment. To install
and Computer RStudio, see: https://rstudio.com.
Configuration
3. For a one-time installation of R packages, use the following:
source("https://bioconductor.org/biocLite.R")
biocLite("xcms") # XCMS
install.packages("data.table") # data.table
install.packages("devtools") # devtools
install.packages(“utils”) # utils
Table 2
XCMS parameters depend on LC/MS instrumentation [16]
Instrument ppm Peakwidth bw mzwid Prefilter

HPLC/Orbitrap 2.5 c(10,60) 5 0.015 c(3,5000)
UPLC/Orbitrap 2.5 c(5,20) 2 0.015 c(3,5000)
HPLC/Q-TOF (high resolution) 15 c(10,60) 5 0.015 c(0,0)
UPLC/Q-TOF (high resolution) 15 c(5,20) 2 0.015 c(0,0)
devtools::install_github("pattilabwu/Credential3.1") #
Credentialing
4. There are several versions of the credentialing software avail-

able. This protocol describes the Credential3.1 package, which
is a development of the original credentialing software
designed to simplify analysis for less experienced users.
4.2 XCMS Parameter Depending on user instrumentation and methods, optimized para-
Settings meters for XCMS may vary [15]. Table 2 provides some sugges-
tions for XCMS settings as previously published [16]. More
information can be found at the link below:https://bioconductor.
org/packages/release/bioc/manuals/xcms/man/xcms.pdf
Acknowledgments
This work was supported by NIH grants R35ES028365 and

R21CA191097 as well as support from the Pew Scholars Program
in the Biomedical Sciences, the Edward Mallinckrodt, Jr., Founda-
tion, and Agilent Technologies.
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Chapter 2
High-Throughput Measure of Bioactive Lipids Using

Non-targeted Mass Spectrometry
Kim A. Lagerborg, Jeramie D. Watrous, Susan Cheng, and Mohit Jain
Abstract
Bioactive lipids represent critical intra- and intercellular signaling molecules, and have been implicated in
both physiologic homeostasis and disease pathology. Measurement of bioactive lipids is vital toward
understanding the role of these signaling intermediates in human biology. Current analytical methods for
assessment of bioactive lipids in human biosamples are limited, however, in breath of analytes assayed as well
as robustness and time required for measures across thousands of samples. Herein, we describe in compre-
hensive detail a rapid and robust analytical method using liquid chromatography-mass spectrometry
(LC-MS) for non-targeted measurement of over 7000 bioactive lipids, including eicosanoids and
eicosanoid-related metabolites, in human biosamples. These methods may be applied to the study of
population scale cohorts to uncover previously unrecognized roles for bioactive lipid species in human
biology.
Key words Bioactive lipids, Eicosanoids, Mass spectrometry, Lipidomics, LC-MS
1 Introduction
Bioactive lipids represent vital intra- and intercellular signaling

molecules that have been linked to cellular function [1]. These
ancient molecules are present in virtually all tissues and are involved
in the modulation of inflammation, immune regulation, and body
homeostasis [2, 3]. Further, lipids have been implicated in a num-
ber of common diseases, including diabetes, cardiovascular disease,
and Alzheimer’s disease [4, 5]. While bioactive lipids represent a
diverse grouping of molecules, the most notable members derive
from 20 carbon arachidonic free fatty acid, and are termed eicosa-
noids [3]. Eicosanoids, in turn, include a number of chemically
Susan Cheng and Mohit Jain contributed equally to this work.
Sarah-Maria Fendt and Sophia Y. Lunt (eds.), Metabolic Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1862,
https://doi.org/10.1007/978-1-4939-8769-6_2, © Springer Science+Business Media, LLC, part of Springer Nature 2019
17
18 Kim A. Lagerborg et al.
related subgroups, including prostaglandins, prostacyclins, throm-

boxanes, and leukotrienes. These agents play an important role as
systemic mediators of inflammation and are the target of common
pharmacologic agents, including aspirin, Cox-2 inhibitors, and
leukotriene receptor antagonists [6, 7]. Given the biological rele-
vance to human health and disease, there is great interest in devel-
opment of analytical approaches for rapid and accurate measure of
bioactive lipids including eicosanoids and eicosanoids related mole-
cules in human biosamples.
Prior analytical tools for measure of bioactive lipids have
relied largely on targeted mass spectrometry based lipidomics
approaches using multiple reaction monitoring. Widely utilized
methods described by Deems et al. [8] allow for the measure-
ment of over 60 eicosanoids in a single analytical run of cells and
media using liquid chromatography coupled to tandem mass
spectrometry (LC-MS/MS) over a 16 min gradient. Other tar-
geted methods have focused on detection of a smaller subset of
10–40 bioactive lipids [9–12]. More recently, methods have been
adapted to increase the breadth of analytes assayed, with current
state-of-the-art LC-MS/MS approaches allowing for the simul-
taneous monitoring of 184 eicosanoids over a 5 min gradient
[13]. Review of the LIPIDMAPS and related chemical databases
[14], however, reveals hundreds to thousands of distinct eicosa-
noids and eicosanoid related metabolites, including those derived
from polyunsaturated fatty acids related to arachidonic acid,
such as eicosapentaenoic acid and dihomo-γ-linolenic acid
[6, 12]. The application of high-resolution mass spectrometry
operating in full-scan “non-targeted” mode now enables measure
of thousands of metabolites simultaneously, with separation
based on accurate mass characteristics. Herein, we describe a
rapid and robust analytical method using liquid chromatography
coupled to high-resolution mass spectrometry for non-targeted
measure of eicosanoids and related bioactive lipids in human
biosamples. All described methods have been optimized for auto-
mated sample and liquid handling for high-throughput applica-
tion. Analysis of 20 μL of human plasma using these methods
allows for relative quantitation of >7000 bioactive lipids, con-
sisting of 235 known eicosanoids, 26 docosanoids, 14 bile acids,
13 endocannabinoids, 11 sterols, 34 free fatty acids, and 17 fatty
acid esters of hydroxy fatty acids (FAHFAs), at physiologic con-
centrations (Fig. 1). These methods will provide new opportu-
nities for discovery of novel bioactive lipid species and their
relation to human biology.
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Ellis’s opinion of the working of “tapu”—muzzled pigs and blindfolded chickens
—tapued pigs—the ceremony of taking “tapu” off the porkers—the princes and
noblemen exhibit their prowess in carrying pigs about—presenting his majesty
with pig’s fry—a tremendous feast—The institution of Tapu in New Zealand—
inconvenience of being tapued—a good jacket lost through the superstition—
The terrible tinder box—how to secure a canoe—the chief’s sacred head—the
sacred kumara grounds—the sacred pole and the missionaries—the chief’s
backbone—the difference between noa and tapu—tapuing a river—the Pakeka
and the iron pot—one of the best uses of tapu—its advantages and
disadvantages—Tapu among the Samoans—snake and shark and thunder
tapus—Born tapus—witchcraft in New Zealand—Introduction of an Englishman
to a “retired” witch—how he found her—she declines to act, having given up the
business and become a “praying” woman—she is persuaded, and sets about
humbugging the visitors—the little beetle in the thatch—the god begging a
blanket—the Englishman not perfectly convinced—Religion among the Dayaks
of the land and sea—the supreme Tewata—extent of their religious system
—“physic” for sacred anointing—the four chief spirits of the Dayak belief—how
man became less than the spirits—what becomes of the Dayak when he dies—
the artful “Umot Perusong”—“Mino Buau,” or warrior ghosts—alarming
apparition of a headless dog—Dayak methods of propitiating the gods—lucky
days, omens, and warnings—the ceremony of making brothers—how
Singauding became the brother of Mr. St. John—a sanguinary cigarette—how
the Kiniahs enter into the bands of brotherhood—making brothers in Western
Africa—pledged in blood—Dayak good and evil spirits—what it is to dream of
Singallong Burong the god of war—delicate way of alluding to small-pox—has
“he” left you?—the Karam of Baram and his god Totadungan—the Dayak who
went to heaven—what he saw—the sacred Bornean bull—Dayak superstitions
concerning dogs and snakes—the legend of the painted dog—the savage of
North America and his “happy hunting ground”—“Kitchi-manitou” and “Matchi-
munedoo”—the North American Indians’ version of the flood—a savage Noah—
how the earth was reformed—the loon, the beaver, and the musk-rat—a legend
of Kitchi-Manitou—he meets the first man—pitying his disconsolate condition he
finds him a mate, Mami by name—the happy meeting—their garden of Eden
with its “tables and chairs and glass windows”—Kitchi-Manitou warns them
against the fruit of the evil tree—Mami is tempted and falls—she invites her
husband and his weakness is too strong for him—anger of Kitchi-Manitou—
banishment of the erring pair—they begin their worldly cares—Mami’s husband
finds a book—finding it too big to carry about he declines to appropriate it—he
is furnished with one of convenient size in which he learns the art of medicine—
the Indian who made a return trip to heaven—how he got there, what he saw,
and how he got back to earth again—the man of the fire-stone—his great
enemy Manabozho—they have a terrible fight and the fire-stone man is beaten
—how Manabozho exerted himself for the good of mankind—his friend
Chibiabos—the Manitous play Chibiabos a cunning trick—grief of Manabozho—
the friends are united—discovering a “thunder’s nest”—Hans Hansom and the
beaver trapper—“second sight” among the Winnebagos—the prophecy—its
verification—the dream of Little Wasp—Indian picture-writing—signs of the
different tribes—what stands for “kill”—a narration in pantomime—Crashey
Jane’s compliments to the editor—Mr. Catlin’s Indian experiences—he paints
the “old bear”—the artist is made much of and likes it—the benighted savages
kiss the hem of Mr. Catlin’s swallow-tail—he does not get on quite so well with
the ladies—all difficulties conquered—Mandan festival of the deluge—“seeking
whom he may devour”—discomfiture and flight of the prince of darkness—
covetousness of the North American Indians—Mr. Murray’s experience on this
head—the old gentleman who coveted the elastic jacket—how he found it a
tight fit—“a good creature in the ice-month”—the Ojibbeway nations of old—the
Pawnees—the Delawares—the confederate six nations—the Shawnees—From
North America to Abyssinia—religious ceremonies observed in that country—
mothers of small account—purification of houses and platters—the Kalijas and
the Lubas—concerning Boudas—Bouda exorcists—Mr. Stern makes the
acquaintance of a Bouda—the woman’s tremendous struggles and arrival of the
exorcist—his operations—he interrogates the Bouda and obtains from it some
interesting information—its strange vagaries before it consents to take its
departure—the Zar—Religion of the Dahoman—the food of the sacred
buzzards—fetish snakes and the punishment for harming them—the Bonny
people and the jewjews—popular superstitions—the king’s charms against
witchcraft—the sacrifice to the bar—the unconscious victim—his doom—sacred
pig’s jaws—“talk and pray”—devil worship—the “Sukia” of the Mosquito shore—
her unprepossessing appearance—Mr. Bard gives her a piece of calico on
condition she stands in the midst of a blazing fire—she earns the calico and
lives to wear it—the belly gods of the Tinguians—Madagascar “the country
without a god”—“Sikidy”—the ceremony of touching the bull—King Peppel’s
religious convictions—a palaver unpalatable to his sable majesty—“Suppose
God were here I must kill him”—a modest wish to live for ever—Mr. Moffat and
the African king—startling news of a universal resurrection—“will all the slain in
battle arise?”—a Namaqua chief’s religious ideas—frying the sun in a pot
—“when we are dead we are dead”—Boles-ki-bo—a Basutos witch-finder—
guessing made easy—end of the farce, commencement of the tragedy—
unclean meats of the Damaras—on the manufacture of rain—drought at
Kuruman—the rain-maker sent for—the commands of the great benefactor—he
churns rain from a milk sack—goat rain and ox rain—the ceremony of the
blighted tree—the sprinkling of the people with a zebra’s tail dipped in water—
still no rain—he demands a baboon “perfect to a hair” and is not sorry that it
cannot be procured—he must have the heart of a lion—he must have
something else too horrible to name, and has it—still the heavens are
inexorable—the last appeal, “It is the face of the white man that scares the rain
clouds”—unpleasant predicament for a man with a white face—the impostor’s
end—A fine day for a butcher’s knife—Figian coming of age—how Mr. Petherick
“Barnumized” the natives as a rain-maker—perpetrates something not many
degrees short of blasphemy—the artful device of the floured flies—a Sabbath in
Equatorial Africa—The worship of Njambai—paying for peeping—“who
bewitched the king?”—the appeal to Ilogo—an unlucky wizard—appalling end of
a witch man—Mfumbo the all-powerful—what came of felling a “devil-tree”—the
business of a Mganga—how he points out the road the traveller should travel—
King Passol’s dancing fetish—his extraordinary performance on stilts—“he be
de debil”—a bal-masqué—“dance, oh snake! for this is indeed a happy day”—
old King Kalabar—“Nabikems”—Kalabar fash—A Yoruba man’s opinion of the
chameleon. Pages 204–336.
PART XII.
SAVAGE DEATH AND BURIAL.
Chap. XXVII.—Disrespect for human life not synonymous with personal
indifference to death—burial ceremonials in cannibal Figi—the Figians no
respecters of persons as regards this custom—preparations for burying a living
king—the “grave grass”—paving the king’s grave—an affectionate son—“see,
his body moves, but it does so unconsciously”—Figian symbols of mourning—
mourning suits of leaves—the “causing to laugh”—murder of the Figian sick
—“pray don’t bury me”—sexton’s work—the poorest savage sure of a
comfortable “narrow bed”—the howling of a dog considered ominous—ditto, a
cat’s clawing on the grave of a woman—how death came into the world—the
sacrifice of fingers—the token of the bloody apron—the art of embalming—the
corpse-praying priest—the “sin hole”—ceremonies at the burial of King Finow—
heroic appeals to the departed king by his warriors—the scene at the sepulchre
—the journey of the sand bearers—shaving the head and burning the cheek
bones—twenty days’ mourning—the Mee too Buggi—singular expression of
fidelity—Finow’s faithful fishermen—the Sandwich Islander’s badge of mourning
—knocking out the teeth—cutting the ears—putting the tongue in black—a
melancholy procession—the house of Keave—a pitiful spectacle—no
admittance to the sacred building—the Pahio tabu—Heathen cities of refuge.
Pages 337–360.
Chap. XXVIII.—Burial rites in Samoa—burying alive—taking his pigs to a better

market—a Samoan inquest—Samoan wakes—carrying a dead body about—
Samoan coffins—dexterous embalming—the mysterious grave fires—a trap to
catch a lost soul— burial customs of the New Zealanders—ornamenting the
dead body—the sexton in Borneo—the weeping-stone of the Permujans—
burning the dead in Western Sarawak—the burning less efficacious than
burying—the hereditary office of sexton—difficulties of finding a sexton—
sepulchral rites of the Sea Dayaks—useful things for consumption in the next
world placed in the grave—Sea Dayaks who fall in battle not disturbed—
Mourning among the Indians of North America—dirtiness the most favourite
symbol—tombs in the air—exorcising an evil spirit—custom of the Sacs and
Foxes—of the Tahkalis—of the New Caledonians—a New Caledonian suttee—
barbarous treatment of the widow—her scorching, and her three years’
mourning and drudgery—the village of the dead—burial unknown among the
Mandans—a Mandan place of skulls—praying to the dead—singular
ceremonies attending the interment of an Ojibbeway—Ojibbeway mourners—
disposing of the property of the dead—a Chippewa ghost story—an invisible
presence—a spirited ghost—veneration for the dead—a royal funeral. Pages
361–385.
Chap. XXIX.—Funeral rites in Damara land—dutiful behaviour of the eldest son of

the deceased—a Damara tomb—offering a pail of milk at the grave—the
Koossan method of disposing of their dead—deserting the sick—duties of the
dead Koossan’s wife—returning in the night to burn down the house—the ox-tail
hair necklace worn by the Koossan widower—Koossan chiefs buried in the
cattle-fold—the magic woman among the Koossans—no recovering spilt water
—no cure, no pay—fate of the unlucky mortal whom the magic woman
denounces—death in Central Africa—waking a defunct man—no half-mourning
among savages—the guests who are invited to the wake—Bota woga—a
tremendous boose—a slave barracoon at Santanga—the sight that M. Chaillu
saw—a thousand bleaching skeletons—funeral ceremonies in Angola—a
jollification in consequence of the death of his mother—the mortal remains of a
Bechuana—planting the top of the head with grass—the burying-ground at
Fetish point—disinclination of the natives to approach the place of graves—the
tomb of old King Passol—a wealthy grave-holder—burying at Anbago, Western
Africa—the bereaved wife carried a pick-a-back—security for “Gungo”—a
Barrodo Beondo funeral—occupying the bed of the deceased—“making a cry”
among the Bulloms and Timannecs—King Archibongo and his devil house—the
painted widows—the “chop-nut” test—Malagasey burial rites—ceremonials
observed on the death of Prince Razahooatrino—lying in state—the attendant
slaves and the fly fanners—subscription among the mourners to pay the funeral
expenses—1500 oxen slain and eaten at a funeral feast—stepping over dead
oxen—no special places for burial in Madagascar—death in Australia—the
name of the dead never mentioned by the surviving relatives—perching dead
old women on tree boughs—“take that for dying!”—the Abyssinian a believer in
the doctrine of purgatory—dancing and singing and face-scratching—funeral of
an Ailat man—how the Sambo Indian is buried—the body in the pitpan—
running away with the corpse to cheat the devil—artful device of the corpse-
bearers—cutting down the palm trees—the way to find out if “Wulasha” has
been cheated—what the traveller Stephens saw at La Rayas, in Mexico—a
Christian burial—death in Dahomey—the very last grand custom—the king’s
ingenious device for the more ready performance of human sacrifice—a victim
saved—how a Dahoman king is buried—providing his majesty with means for
paying his way in the next world. Pages 386–418.
List of Wood-Cuts,
FROM DESIGNS BY HARDEN S. MELVILLE.
ENGRAVED BY H. NEWSOM WOODS.
PAGE
FORBE’S RECEPTION BY THE KING OF DAHOMEY 1
A MALAGASEY BALL 52
BORNEO 54
AUSTRALIAN WEAPONS 66
POLYNESIAN WAR CANOE 67
WAR DANCE OF NEW ZEALANDERS 88
DAYAK AND MALAY WEAPONS 93
POLYNESIAN GOODS BOAT 106
NORTH AMERICAN WAR WEAPONS 111
A CHIPPEWA WARRIOR 115
POLYNESIAN WAR TOOLS 122
THE EUROPEAN’S HUT IN THE WILDERNESS 133
TORRES’ STRAITS CANOE 147
AN AUSTRALIAN DUEL 154
AFRICAN ARMS 162
THE UNIVERSAL WEAPON 168
A SAVAGE BOWMAN 169
PAPUAN BLACKSMITHS 180
THE EXPLORER’S HIGHWAY 183
THE TWO DOGS OR NONE 192
BOATMEN OF ROCKINGHAM BAY 203
THE TRUE WORD EXPOUNDED IN WESTERN AFRICA 204
SAMOAN IDOL WORSHIP 218
A POLYNESIAN IDOL 221
SPECTRE OF A HEADLESS DOG 240
MAKING BROTHERS 243
THE COVETOUS PAWNEE 275
AN IROQUOIS WARRIOR 280
A WOMAN UNDER THE INFLUENCE OF BOUDA 289
PUNISHMENT FOR KILLING FETISH SNAKES 293
CEREMONY OF TOUCHING THE BULL 301
DIVINATION SCENE 307
MAKING RAIN 312
DU CHAILLU’S PEEP INTO A HEATHEN TEMPLE 321
THE WIZARD IN THE STOCKS 324
INHABITANTS OF THE FAN COUNTRY 336
BURYING ALIVE IN FIGI 337
MOURNING SUIT OF LEAVES 341
FUNERAL OBSEQUIES OF KING FINOW 354
A SAMOAN SEPULCHRE 364
A MANDAN CHIEF 374
MANDAN PLACE OF SKULLS 375
“HE HEARD THEM RECOUNT THEIR VALIANT DEEDS” 380
DAMARA TOMB 387
AFRICAN WAKE 392
THE “MASTER OF LIFE” IN EQUATORIAL AFRICA 395
THE VERY LAST DAHOMAN “CUSTOM” 414
Forbes’s Reception by the King of Dahomey.
PART VII.
SAVAGE KINGS AND COURTS.
CHAPTER XVII.
The Savage considered as a child of nature—A saltatory welcome—
Gezo, King of Kings—Items of Dahoman royal treasure—
Distribution of the presents—Kings and Ambassadors joining in
the scramble—The human sacrifices—A “Grand Custom” of the
year 1862—The King of Abó—The terrible Neam Nam—
Browowdi, King of Issapoo—A King of Old Kalabar—King Eyo
Honesty—The order of Egbo—The Mambo of Lunda—The Jaga.
t first sight it would seem hard to show a greater

anomaly than an unthinking instinct-obeying nation of
savages consenting to be controlled and governed by a
fellow barbarian, equally unthinking, and morally
powerless; and the said anomaly is the more striking
when the savage is viewed as the vulgar view him,—as a free-born
“child of nature,” intolerant of rule, and guided in all his behaviour by
certain instinctive high-souled sentiments, and vast powers of mind,
that require only cultivation to fit their possessor for the achievement
of all that ever was yet successfully attempted by man. This,
however, is very far from the fact. Without doubt, and as we have
only to refer back to our own ancient barbarism to be convinced, the
germ of perfect manhood lies in every savage, but like the ore of
gold and iron, the true metal lies deep, and to free it from dross and
make its lustre apparent is a process neither easy nor rapid. Again,
like golden ore, in which the precious deposit shows here and there
with a sheen that undoubtedly reveals its presence, does the
savage’s mind manifest its existence in fitful flashes and
glimmerings, that, alas! only reveal to him what a helpless wretch he
is, and what a terribly responsible thing is life, with children and wife,
and all its other precious belongings, and which, in an instant, may
be spilt and vanish like a capsized gourd of water.
This—the end of life—is the end of everything with our brother the
savage; life to him is only good according to the ease it enables him
to get in the land he lives in. The first business of his life is to make
himself comfortable; the second is how to hold such appurtenances
to his comfort as he has gained. If he is a little man, any man a trifle
bigger coming his way may strip him, seize his wife and children as
slaves, knock him on the head, and appropriate his hut; if he is a big
man any two big men who choose to conspire may serve him in the
same cruel way: what then remains to be done, but to combine for
the good of the common weal? which may be aptly likened to a
common wheel—the chief being the stock, the various headmen, or
councillors, the spokes, or spokesmen, and the fellowes, just as
many savage fellows as the tribe, or band, or tire embraces.
Still, who is to be “king,” or “chief,” or “Jaga,” or “Mambo,” or
whatever else you please, as representing the stock or common
centre of the said wheel? About this question, however, we need not
trouble ourselves, and simply because, just as the queen bee is born
in a hive, so are men born commanders of men; that is, originally;
the fact of their descendants degenerating, and being totally unfit to
wield a sceptre is nothing to the purpose. Custom and Fashion then
step in, and these two of themselves are monarchs potent enough to
settle the gravest question that could possibly arise, even in the most
civilized countries in the world. Wherever a leader is wanted, a
leader will be found; he may be a wrong-headed leader, or
conceited, or cruel, or arbitrary; but so sure as he remains at the
helm, for the short space only of a year, you may depend that he is
no make-believe; and the very worst you can say of such an one is,
that it is a pity that a king should possess so many bad qualities; that
he deserves to die for them, if you please; nay, go as far as killing
him, and how different are your feelings than though you had killed a
merely contemptible upstart.
Of course I talk of “killing” as a figure of speech, in its extremest
sense. There, however, is one king now existing whom, if with his life
would end the hideous work of blood and carnage prevailing in his
nation, might well be wished dead. I allude to the King of Dahomey,
who, as a trafficker in human beings, dead and alive, is an ulcer on
the face of the world; a man whose guilt is so black that it may never
be washed away, though they laved him in rivers of water as deep as
those of tears and blood that he has caused to flow.
We hear very little of this potentate. Now and then an adventurous
European will penetrate his awful domains, and give to the world
some account of the horrors he sees and hears; once in a while we
read in the African News that “the King of Dahomey threatens a
massacre on such or such a place,” or that the barbarous “annual
custom” is about to commence, with an enumeration of the victims
already secured, and whose blood is required to “water the late
king’s grave.” Of all Englishmen who have witnessed the
abominations of Dahomey, none have recorded them more
graphically than Commander Forbes, and it is from his account
chiefly that what is here related of Dahomey is derived.
Commander Forbes’s first introduction to the King of Dahomey
was, to say the least, calculated to make a lasting impression on his
memory. Within a short distance of the royal residence Mr. Forbes
and his party halted at the house of a friend, and attired themselves
in full uniforms, and then moved forward to some shady trees to
await the arrival of the carbooceers who were to conduct them to the
royal presence. After the adventurous Europeans came a crowd of
hammock-men and other Dahoman followers. About a quarter of a
mile from the halting place stood a vast assembly of carbooceers
and soldiers with umbrellas of state, flat-topped and ornamented like
those of the Chinese, and banners of every hue and most varied
devices. Beside the Dahoman standards, each of which was
ornamented by a human skull, floated the national flags of France,
England, Portugal, and Brazil, whilst every carbooceer had his own
particular pennon.
The first chief who advanced towards Commander Forbes’s party
from this gay crowd of carbooceers was Boh-peh, the governor of
the capital, dressed in a country cloth wrapped round his body, a
slouched hat, necklaces of coral and other beads, and armed with a
handsome sword. Behind him came a retinue of soldiers, his
standard, his umbrella of state, and his stool of rank; and, lastly, a
band of most discordant music. Arriving in front of Forbes’s party, he
bowed, and then marched from right to left round their seats three
times, completing each circuit with a low obeisance. On his third
round he discharged three muskets, and danced a short measure,
then advanced and shook hands, and seated himself on his stool of
office, which its bearer had placed on the Englishman’s right hand.
Ah-hoh-peh, the king’s brother, and Gaseh-doh, the chief of the
carbooceers of Dahomey, followed, with similar attendants and
ceremonies. When the whole party were seated, a body of the royal
household, having half their heads shaved, took position in front, and
sang a hymn of welcome to the Englishmen. The Dahoman guard
were showily dressed in scarlet, trimmed with beads and other
ornaments, with their heads covered by silver caps, some of which
were distinguished by a pair of small silver horns. In his right hand,
each carried a horse-tail whip, with which he beat time to the air of
the chant. Next advanced Poh-neh-soo (at once a military officer,
court-fool, and headsman) and his party of blunderbuss men, who
likewise fired a salute, and then drank healths with the Europeans;
after which, the latter entered their hammocks, and the entire party
proceeded towards the palace, amid the firing of muskets and short
brass guns.
The travellers found the palace of Dange-lah-cordeh surrounded
at a distance of twenty feet with human skulls, many of which had
crumbled with time, or had blown down. The square of the palace
was filled with armed people sitting on their hams, the polished
barrels of their muskets standing up like a forest. Under a thatched
gateway sat the king surrounded by his immediate wives; while on
each side sat the amazons all in uniform, armed and accoutred; and
in the centre of the square squatted the males. Hundreds of banners
and umbrellas enlivened the scene, and a constant firing from great
guns and small arms increased the excitement.
When near the king’s seat, the European party came to a halt,
while the carbooceers bowed down and kissed the dust. Passing
before the throne, they bowed and made the circuit of the square
three times, the carbooceers prostrating themselves each time. Then
the Englishmen stept from their hammocks and approached the king,
who had been reclining, but now rose, and several discordant bands
struck up a quick step, whilst guns were fired, and all shouted,
except the ministers and carbooceers, who prostrated and threw
dust over their heads, as Mr. Forbes advanced and shook hands
with the king.
King Gézo, of Dahomey, was about forty-eight years of age, good
looking, with nothing of the negro feature, and his face wanting
several shades of being black; his appearance was commanding,
and his countenance intellectual, though stern in the extreme.
Indeed, he is described as being short of positively handsome only
by a slight squint. He was plainly dressed in a loose robe of yellow
silk, slashed with satin stars, and half moons, Mandingo sandals,
and a Spanish hat trimmed with gold lace.
Taking their seats facing the royal mat, the party entered into a
complimentary conversation, after which the ministers were
introduced by name to our countrymen. His Majesty then enquired if
his guests would like to see a review of his amazons, and of course
his guests were delighted at the offer. Three regiments were
paraded, one being distinguished by a white cap ornamented with
the blue alligator, another by a blue cross, and the third by a blue
crown. The officers were recognized by their coral necklaces and
superior dresses; while each carried a small whip which they freely
plied when required. Firing, rushing hither and thither, and advancing
to the throne to address the king, were the chief features of the
review; at the conclusion of which two amazon heralds, bearing long
trumpets, blew a blast and then blazoned forth the numerous names
of Gézo, King of Kings.
The king having asked Commander Forbes to drink, rose, and
with his glass in hand tapped that of each of his guests; then there
thundered forth a salute of guns almost drowned by the shouts of the
multitude. The ministers and carbooceers danced, and the ladies
held clothes before the king. Men must not see the king eat or drink.
On the whole it was Mr. Forbes’ distinct conviction that no king could
have been more civil or more condescending.
The same gentleman had the good (?) fortune to be present at the
ceremony of Ek-bah-tong-ek-bah, or “display of the king’s wealth,”
an exhibition of a perfectly unique character and finding no parallel
throughout the world. The fundamental principle of the King of
Dahomey’s government is profuse generosity to his subjects. His
constant aim is to inculcate the notion that his riches are boundless
and his good nature none the less so. How hollow and fictitious are
both these assumptions was evident enough to Commander Forbes,
although for his head’s sake he dare not express such a conviction
while in the land of “Grand Customs.”
“It was little more than seven o’clock a.m. when we were informed
that a royal messenger had arrived to summon us to witness the
custom to be performed on this day—the Ek-bah-tong-ek-bah, or
“display of the king’s wealth.” At a little distance from our gate the
road was fenced off and a guard set on the temporary gate, so as to
prevent any one entering who was not invited to bear a part in the
proceedings of the day. They who wished to inspect the royal
treasures which were to be shown to the people assembled in the
Ahjahee market-place.
“When we arrived at the palace square at the foot of the ladder
leading to the palace house, on each side were three human heads
recently decapitated, the blood still oozing; on the threshold of the
entrance gate was a pool of blood from six human sacrifices over
which we had to step. In the square was a huge model of an
elephant caparisoned on wheels, on which the king is drawn when
going short journeys. The king never walks, nor rides on horseback,
but is either carried in a hammock, or drawn on this elephant, or in a
carriage or wheeled chair. In the centre of the court-yard stood a
crimson tent or pavilion forty feet high, ornamented with emblems of
human and bullock’s heads, skulls, and other devices equally
barbarous and disgusting. On the top was the figure of a Dahoman
standard-bearer (or half-heads, as they are called, having half their
heads shaved) bearing a standard, having for a device a skull in a
calabash standing on three other skulls. About the yard were many
flags of all colours, some having as their devices men cutting off
other’s heads, and others tying prisoners, and many national flags,
amongst which were several Union Jacks. In and about the pavilion
were the female host of ministers, carbooceers, amazons, wives,
and virgins. The king had not arrived; all were gaily dressed, and
armed, and accoutred.
“On the neutral ground where we stood facing the pavilion (while
the mayo and ce-a-boo-gan grovelled in the dust like mandarins
kow-towing to the royal chair) roamed an ostrich, an emu, several
dwarfs, hunchbacks, and albinoes, besides troops of dogs of almost
every country and variety. All the ministers and carbooceers were
arrayed in red-striped flowing robes laden with necklaces of coral
and other beads. Each wore a scimitar, a short sword, and a club.
“Presently, under a salute fired from musketoons and small brass
pieces within the court and cannon outside, the king arrived, dressed
in a white silk flowing robe flowered in blue and a gold-laced hat, and
took his seat in a sofa under the pavilion. Forthwith the bands struck
up and the heralds proclaimed that Gézo, the Leopard and the
Hawk, had taken his place; fifty-eight ministers and carbooceers at
the same time marched three times in single file, and at the third
time all prostrated and kissed the dust. So soon as this ceremony
was concluded the business of the day commenced. This is a public
display of the monarch’s wealth, carried on the heads of slaves
through the town to the market and back again. The procession
consisted of between six and seven thousand people.”
To enumerate, however, every item of “wealth” carried by these
six or seven thousand individuals would certainly be to weary the
reader, even though she were a lady, loving, next to possessing gold
and gems, to hear and read about them; besides, there is much
among the Dahomey “crown jewels” which the said lady reader could
match in point of value in her wash-house or lumber-room. Let us
take a few notes of the members of the procession:—
52 women carrying white flowered vases.
6 carrying jars.
10 carrying French ornaments under glass shades.
1 carrying a washing pan.
1 carrying a crimson cushioned rocking chair.
1 carrying a box.
1 carrying a washing-stand.
1 carrying a toilette table, drawers, and glass.
2 carrying stools.
3 carrying banners.
1 carrying a skull in a copper pan.
2 carrying calabashes full of skulls.
2 carrying shields.
Head bunseh’s mother in scarlet, wearing a Life Guardsman’s

helmet and plumes, and attended by a lady in Charles II. hat and
plumes, both magnificently dressed.
8 Malam’s wives.
Band of 20.
Guard of 100.
Band of 12.
4 women carrying pans of skulls.
2 carrying jars surmounted with skulls.
1 carrying a large pan of skulls.
1 carrying a banner.
2 carrying umbrellas over the king’s women and attendants, in
crimson cloth dresses and slouched hats trimmed with gold.
Band of 20.
Guard of 30.
2 women carrying pans of skulls.
2 carrying jars of skulls.
2 carrying a banner and two umbrellas each.
King’s grandmother, in head-dress of silver, crimson and silver
robe and train, held by a maiden bearing a gold-headed stick.
One of the King’s grandfather’s widows in scarlet and gold.
1 man carrying a banner.
1 carrying a tray containing three human skulls.
The King’s washing-tub borne by 30 guards.
2 men carrying a scarlet and gold sedan chair.
300 carrying dishes with a basket in each.
55 carrying blue glass goblets.
50 carrying white glass goblets.
6 carrying a drum trimmed with skulls.
1 carrying umbrella ornamented with eighty human jaw-bones.
Men carrying a native sofa. Etc. etc. etc.
All the possessions of the king, in fact, from his grandmother to
his washing-tub, were to be found, and made no doubt as a whole a
tremendous display, though it is by no means unreasonable to say
that the sum of the “king’s wealth” brought under the hammer of a
London auctioneer would realize little more than would the contents
of any first-rate villa-residence at Clapham or Richmond.
It should be stated, however, that as well as this household gear,
the royal exchequer was brought out and carried in measures. In this
again the king was fortunate as regards opportunity for display;
cowries form the currency of Dahomey—and goodness knows the
many thousands of these it takes to make a single English sovereign
—therefore it was easy enough to arrange that, although in form of
money the king possessed no more than a little over a thousand
pounds, porter after porter should go trooping past, each with such a
load of money as made the mouths of the spectators water with
envy.
Well, their longing was not to remain entirely ungratified. The king,
although a vastly rich, is not a greedy man; and annually he makes
presents to one and all of his loyal subjects; not in a hole and corner
sort of way, but publicly—with the mob before him, and the riches,
the cowries, the bales of cloth, the tobacco, and the kegs of rum in
heaps and piles and pyramids at his elbow; and scrambles the
astonishing gifts fairly and without favour—or so it seems. Let us,
however, see what Mr. Forbes has to say on the subject.
“On the last day of May, commenced the custom of the Ek-que-
noo-ah-toh-meh, or throwing the presents from the Ah-toh. It is on
this day human sacrifices are offered by the king among his gifts to
his people. In the centre of the market-place a platform was erected
twelve feet in height, enclosed by a parapet breast high. The whole
was covered with cloths of all colours, and surmounted by tents,
gaudy umbrellas, and banners of varied hues and devices, among
which, as usual, were several Union Jacks. On the west front of the
Ah-toh, which must have been at least one hundred feet square, was
a barrier of the prickly acacia, and within this were the victims for the
day’s sacrifice, lashed in baskets and canoes as on yesterday. A
dense naked mob occupied the area, whilst a guard of soldiers
prevented them from bearing down the barrier. Beyond, in all
directions, were groups of people collected round the banners and
umbrellas of the different ministers and cabooceers. The naked mob
consisted of the soldiers of the king, his brothers and sons, the
ministers and higher cabooceers: each carried a grass-cloth bag
round his waist; and the actual business of the day was a public
display of the generosity of the king, who scrambles goods of all
kinds among these warriors.
“The king had preceded us, and as we took our seats under a
canopy to the right of the Ah-toh, His Majesty appeared on the
platform under the shade of a handsome umbrella of crimson velvet
and gold, dressed in an old black waistcoat, a white nightcap, and a
cloth round his loins, and was greeted with loud shouts from the
military expectants, who now formed into bands, and carrying their
officers on their shoulders, marched past the royal position, the
king’s own taking the lead. This they did three times, and then halted
immediately under the king’s position, who harangued them on the
impropriety of fighting during the scramble, and having thrown a few
cowries by way of trial, commanded us to join him.
“Ascending the ladder the appearance was truly novel: in three
separate heaps in different parts of the platform were three thousand
heads of cowries, several heaps of cloths, rum in kegs, and rolls of
tobacco; one side was occupied by tents for the royal wives; while
others were grouped about in different parts of the platform in gaudy
dresses. At the upper end stood the king surrounded by his
ministers, and at the lower were, under canopies of showy
umbrellas, two tables bearing liquors and glasses, one for the cha-
cha, the other for ourselves. After taking our seats we were directed
to stand under an umbrella facing the mob, and now commenced in
real earnest the scramble, the king labouring hard throwing down
cowries, cloth, tobacco, etc. The cowries appeared to be the
property of the lucky ones who caught them, but the cloths were
instantly handed to the officers, and if not, a fight ensued that was
terrible to behold.
“The naked multitude emitted an effluvium only to be compared to
the fetid atmosphere of a slave ship, and as the mass oscillated,
there arose a vapour like the miasma of a swamp, as they were
perfectly bathed with perspiration.
“Besides throwing gifts to the soldiers, His Majesty was all smiles
and liberality in his donations to the ministers and a number of
others; but to no one was any large sum given. At one time he sent
us a basket containing ten heads of cowries and two pieces of cloth
as a present, and at another a constant supply of cowries and cloths
to scramble among the mob.
“Among the recipients of the royal bounty were two kings and
several ambassadors, including one from Ashantee called Cocoa
Sautee.
“Towards noon the brigantine on wheels put off to discharge her
cargo of rum, tobacco, and cowries, which were added to the heaps
on the platform. The king’s party of soldiers keeping together, were
evidently the principal recipients, and we soon found that something
like an equal distribution among them was aimed at. A captain of
musquetoon-men, named Poh-veh-soh, at once a military officer,
court fool, and headsman, caught my attention, and I threw him three
pieces of cloth full of cowries; on receiving the third he was ordered
off the ground. Rum was distributed to the élite on the platform, and
a breakfast provided for us, besides food for the ministers and wives.
“By two o’clock one of the heaps of one thousand heads of
cowries had been thrown away and part of another given to the
higher classes. Some three or four hundred pieces of cloth, a few
kegs of rum, and rolls of tobacco, having all disappeared, His
Majesty retired to rest awhile.
“Would to God that I could here close the account of this day’s
proceedings, simply detailing the barbarous policy of raising the
worst passions of man in order to make people believe in the profuse
distribution of a pay which if doled out individually would be a mere
pittance. The crowd can have no idea of the sum scrambled for; all
they know is that a continuous shower is kept up for seven hours,
and they consider it must be immense. Even if a man gets none he
is content to know that he has been unfortunate, and should he
proclaim his ill-luck he would not be believed, each supposing the
other to be disguising the real quantity he has gained.
“During the royal absence a dead silence reigned as if by general
consent; when by accident it was broken it was reinforced by the
eunuchs sounding their metal bells, tolling the knell of eleven human
beings. Out of fourteen now brought on the platform, we, the
unworthy instruments of the Divine will, succeeded in saving the
lives of three. Lashed in their baskets these sturdy men met the gaze
of their persecutors with a firmness perfectly astonishing. Not a sigh
was breathed. In all my life I never saw such coolness so near death.
It did not seem real, yet it soon proved frightfully so. One monster
placed his finger to the eyes of a victim who hung down his head, but
finding no moisture drew upon himself the ridicule of his fiendish
coadjutors. Ten of the human offerings to the bloodthirsty mob, and
an alligator, and a cat, were guarded by soldiers, the other four by
amazons.
“In the meantime the king returned, and calling us from our seats
at the further end of the platform, asked if we should wish to witness
the sacrifice. With horror we declined, and begged to be allowed to
save a portion of them. After some conversation with his courtiers,
seeing him wavering, I offered him a hundred dollars each for the
first and last of the ten, while at the same time Mr. Beecroft made a
similar offer for the first of the four, which was accepted, and the
three were immediately unlashed from their precarious position, but
forced to remain spectators of the horrid deed to be done on their
less fortunate countrymen. What must have been their thoughts?

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Metabolic Signaling: Methods and

Protocols Sarah-Maria Fendt

Systems Metabolic Engineering Methods and Protocols 1st

TGF β Signaling Methods and Protocols 1st Edition Xin-

Yeast Metabolic Engineering Methods and Protocols 1st

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Suppression and Regulation of Immune Responses Methods

Zebrafish Methods and Protocols Koichi Kawakami

SNAREs: Methods and Protocols Rutilio Fratti

Epitranscriptomics: Methods and Protocols Narendra

For further volumes:

Methods and Protocols

ISSN 1064-3745 ISSN 1940-6029 (electronic)

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Leuven, Belgium Sarah-Maria Fendt

14 Assessing the Impact of the Nutrient Microenvironment

ABDIEL ALVARADO-DIAZ  Laboratory of Exercise and Health, Department of Health Sciences

JUAN FERNÁNDEZ-GARCÍA  Laboratory of Cellular Metabolism and Metabolic Regulation,

OLIVER D. K. MADDOCKS  Wolfson Wohl Cancer Research Centre, Institute of Cancer

JERAMIE D. WATROUS  Department of Medicine, University of California, San Diego,

A Protocol to Compare Methods for Untargeted

Key words Untargeted metabolomics, Metabolite profiling, Metabolism, Credentialing, Liquid

Metabolite profiling, or metabolomics, can be performed with

however, simple optimization of untargeted metabolomic methods

The following protocol consists of two main parts. First, we

2.1 Purchasing Metabolic extracts of credentialed E. coli (strain K12, MG1655)

2.2 Media 1. 5 M9 minimal salts. Final concentration: 33 g/L disodium

8. 250 mL media filter with a 0.22 μm pore size.

2.4 Credentialed 1. Two 10 mL beakers.

2.5 Credentialed 1. Two 50 mL conical tubes.

2.6 LC/MS 1. LC/MS-grade solvents (e.g., water, acetonitrile, methanol,

3.2 Initial E. coli 1. We recommend performing this portion of the experiment by

5. To the “13C glucose” flask, add 207 mg of uniformly labeled

7. Rinse the top of the pellet with 0.5 mL of ultrapure water by

3.6 Credentialed 1. Perform the metabolite extraction protocol of choice (for

3.7 LC/MS Data Credentialing is compatible with most data-processing software, as

raw LC/MS data Stage 1: LC/MS Data Collection

.mzXML files other data processing pipelines

Fig. 1 Three stages of the credentialing workflow

3. A detailed template is available online at the link below to

2. Use one of the following to set up a working directory, depend-

3. Note that every time a new R session is opened, all required

4. If you are using a data-processing pipeline that does not rely

3.7.2 Data Processing 1. Open MSConvert.

Fig. 2 Screenshot illustrating the conversion of raw MS data with MSConvert

retention time correction, and missing peak filling. Run the

Automated Processing xsGroupExtract() is a function in the Credential3.1 R package. It

*Use the help(xsGroupExtract) command to see the description of

Fig. 3 Screenshot of a CSV file with appropriate format for credentialing

include only four columns: “cc”, “mz”, “rt,” and “i”. An

3.7.4 Credentialing Run the following to perform credentialing:

credential = credentialing(features1T1,features1T2,ppm = 20,

*Parameters of the credentialing() function:

4.1 R Initiation, 1. To install R, see: https://cran.r-project.org/.

Instrument ppm Peakwidth bw mzwid Prefilter

4. There are several versions of the credentialing software avail-

This work was supported by NIH grants R35ES028365 and

(19):10397–10406. https://doi.org/10. 12. Naser FJ, Mahieu NG, Wang L et al (2018)

High-Throughput Measure of Bioactive Lipids Using

Key words Bioactive lipids, Eicosanoids, Mass spectrometry, Lipidomics, LC-MS

Bioactive lipids represent vital intra- and intercellular signaling

Susan Cheng and Mohit Jain contributed equally to this work.

related subgroups, including prostaglandins, prostacyclins, throm-

ABDIEL ALVARADO-DIAZ Laboratory of Exercise and Health, Department of Health Sciences

JUAN FERNÁNDEZ-GARCÍA Laboratory of Cellular Metabolism and Metabolic Regulation,

OLIVER D. K. MADDOCKS Wolfson Wohl Cancer Research Centre, Institute of Cancer

JERAMIE D. WATROUS Department of Medicine, University of California, San Diego,