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METABOLOME ANALYSIS
METABOLOME ANALYSIS
An Introduction

SILAS G. VILLAS-BÔAS
AgResearch Limited
Grasslands Research Centre
New Zealand

UTE ROESSNER
Australian Centre for Plant Functional Genomics
School of Botany, University of Melbourne, Australia

MICHAEL A. E. HANSEN
JORN SMEDSGAARD
JENS NIELSEN
Center for Microbial Biotechnology, BioCentrum-DTU
Technical University of Denmark
Copyright © 2007 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New Jersey

Published simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data:

Metabolome analysis : an introduction / Silas G. Villas-Bôas … [et al.].
p. ; cm.
Includes bibliographical references.
ISBN-13: 978-0-471-74344-6
1. Metabolites. 2. Genomics. I. Villas-Bôas, Silas G. (Silas Granato)
[DNLM: 1. Metabolism. 2. Cell Physiology. 3. Genomics–methods.
4. Systems Biology–methods. QU 120 M587973 2007]
QP171.M48 2007
572.8’6–dc22 2006022114

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1
 To
our colleagues,
families and
friends
CONTENTS

PREFACE xiii

LIST OF CONTRIBUTORS xv

PART I: CONCEPTS AND METHODOLOGY

1 Metabolomics in Functional Genomics and Systems Biology 3

1.1 From genomic sequencing to functional genomics, 3
1.2 Systems biology and metabolic models, 6
1.3 Metabolomics, 8
1.4 Future perspectives, 11

2 The Chemical Challenge of the Metabolome 15

2.1 Metabolites and metabolism, 15
2.2 The structural diversity of metabolites, 18
2.2.1 The chemical and physical properties, 18
2.2.2 Metabolite abundance, 23
2.2.3 Primary and secondary metabolism, 24
2.3 The number of metabolites in a biological system, 25
2.4 Controlling rates and levels, 26
2.4.1 Control by substrate level, 27
2.4.2 Feedback and feedforward control, 27

vii
viii CONTENTS

2.4.3 Control by “pathway independent” regulatory molecules, 27

2.4.4 Allosteric control, 28
2.4.5 Control by compartmentalization, 30
2.4.6 The dynamics of the metabolism—the mass flow, 31
2.4.7 Control by hormones, 33
2.5 Metabolic channeling or metabolons, 33
2.6 Metabolites are arranged in networks that are part of a cellular
interactome, 35

3 Sampling and Sample Preparation 39

3.1 Introduction, 39
3.2 Quenching—the first step, 41
3.2.1 Overview on metabolite turnover, 41
3.2.2 Different methods for quenching, 44
3.2.3 Quenching microbial and cell cultures, 44
3.2.4 Quenching plant and animal tissues, 50
3.3 Obtaining metabolites from biological samples, 52
3.3.1 Release of intracellular metabolites, 52
3.3.2 Structure of the cell envelopes—the main barrier
to be broken, 52
3.3.3 Cell disruption methods, 58
3.3.4 Nonmechanical disruption of cell envelopes, 59
3.3.5 Mechanical disruption of cell envelopes, 66
3.4 Metabolites in the extracellular medium, 71
3.4.1 Metabolites in solution, 72
3.4.2 Metabolites in the gas phase, 75
3.5 Improving detection via sample concentration, 76

4 Analytical Tools 83
4.1 Introduction, 83
4.2 Choosing a methodology, 84
4.3 Starting point—samples, 86
4.4 Principles of chromatography, 87
4.4.1 Basics of chromatography, 87
4.4.2 The chromatogram and terms in chromatography, 90
4.5 Chromatographic systems, 93
4.5.1 Gas chromatography, 94
4.5.2 HPLC systems, 102
4.6 Mass spectrometry, 106
4.6.1 The mass spectrometer—an overview, 107
4.6.2 GC-MS—the EI ion source, 109
4.6.3 LC-MS—the ESI ion source, 111
4.6.4 Mass analyzer—the quadrupole, 115
4.6.5 Mass analyzer—the ion-trap, 117
CONTENTS ix

4.6.6 Mass analyzer—the time-of-flight, 119

4.6.7 Detection and computing in MS, 121
4.7 The analytical work-flow, 125
4.7.1 Separation by chromatography, 125
4.7.2 Mass spectrometry, 128
4.7.3 General analytical considerations, 129
4.8 Data evaluation, 129
4.8.1 Structure of data, 129
4.8.2 The chromatographic separation, 132
4.8.3 Mass spectral data, 133
4.8.4 Exporting data for processing, 135
4.9 Beyond the core methods, 136
4.9.1 Developments in chromatography, 137
4.9.2 Capillary electrophoresis, 139
4.9.3 Tandem MS and advanced scanning techniques, 141
4.9.4 NMR spectrometry, 143
4.10 Further reading, 144

5 Data Analysis 146

5.1 Organizing the data, 146
5.2 Scales of measurement, 147
5.2.1 Qualitative data, 148
5.2.2 Quantitative data, 148
5.3 Data structures, 148
5.4 Preprocessing of data, 150
5.4.1 Calibration of data, 150
5.4.2 Combining profile scans, 151
5.4.3 Filtering, 152
5.4.4 Centroid calculation, 156
5.4.5 Internal mass scale correction, 156
5.4.6 Binning, 157
5.4.7 Baseline correction, 157
5.4.8 Chromatographic profile matching, 163
5.5 Deconvolution of spectroscopic data, 166
5.6 Data standardization (normalization), 167
5.7 Data transformations, 168
5.7.1 Principal component analysis, 168
5.7.2 Fisher discriminant analysis, 171
5.8 Similarities and distances between data, 173
5.8.1 Continuous functions, 173
5.8.2 Binary functions, 176
5.9 Clustering techniques, 178
5.9.1 Hierarchical clustering, 178
5.9.2 k-means clustering, 181
x CONTENTS

5.10 Classification techniques, 182

5.10.1 Decision theory, 183
5.10.2 k-nearest neighbor, 184
5.10.3 Tree-based classification, 184
5.11 Integrated tools for automation, libraries, and data evaluation, 185

PART II—CASE STUDIES AND REVIEWS

6 Yeast Metabolomics: The Discovery of New Metabolic Pathways in

Saccharomyces cerevisiae 191
6.1 Introduction, 191
6.2 Brief description of the methodology used, 192
6.2.1 Sample preparation, 192
6.2.2 The analysis, 194
6.3 Early discoveries, 194
6.4 Yeast stress response gives evidence of alternative pathway for glyoxylate
biosynthesis in S. cerevisiae, 195
6.5 Biosynthesis of glyoxylate from glycine in S. cerevisiae, 196
6.5.1 Stable isotope labeling experiment to investigate glycine catabolism
in S. cerevisiae, 198
6.5.2 Data leveraged for speculation, 201

7 Microbial Metabolomics: Rapid Sampling Techniques to Investigate

Intracellular Metabolite Dynamics—An Overview 203
7.1 Introduction, 203
7.2 Starting with a simple sampling device proposed by Theobald
et al. (1993), 204
7.3 An improved device reported by Lange et al. (2001), 205
7.4 Sampling tube device by Weuster-Botz (1997), 207
7.5 Fully automated device by Schaefer et al. (1999), 209
7.6 The stopped-flow technique by Buziol et al. (2002), 209
7.7 The BioScope: a system for continuous-pulse experiments, 212
7.8 Conclusions and perspectives, 213

8 Plant Metabolomics 215

8.1 Introduction, 215
8.2 History of plant metabolomics, 217
8.3 Plants, their metabolism and metabolomics, 219
8.3.1 Plant structures, 219
8.3.2 Plant metabolism, 222
8.4 Specific challenges in plant metabolomics, 223
8.4.1 Light dependency of plant metabolism, 223
CONTENTS xi

8.4.2 Extraction of plant metabolites, 225

8.4.3 Many cell types in one tissue, 225
8.4.4 The dynamical range of plant metabolites, 226
8.4.5 Complexity of the plant metabolome, 226
8.4.6 Development of databases for metabolomics-derived data in plant
science, 228
8.5 Applications of metabolomics approaches in plant research, 229
8.5.1 Phenotyping, 229
8.5.2 Functional genomics, 231
8.5.3 Fluxomics, 232
8.5.4 Metabolic trait analysis, 232
8.5.5 Systems biology, 234
8.6 Future perspectives, 234

9 Mass Profiling of Fungal Extract from Penicillium Species 239

9.1 Introduction, 239
9.2 Methodology for screening of fungi by DiMS, 242
9.2.1 Cultures, 243
9.2.2 Extraction, 243
9.2.3 Analysis by direct infusion mass spectrometry, 244
9.3 Discussion, 245
9.3.1 Initial data processing, 245
9.3.2 Metabolite prediction, 246
9.3.3 Chemical diversity and similarity, 248
9.4 Conclusion, 252

10 Metabolomics in Humans and Other Mammals 253

10.1 Introduction, 253
10.2 A brief history of mammalian metabolomics, 257
10.3 Sample preparation for mammalian metabolomics studies, 260
10.3.1 Working with blood, 262
10.3.2 Working with urine, 263
10.3.3 Working with cerebrospinal fluid, 264
10.3.4 Working with cells and tissues, 267
10.4 Sample analysis, 268
10.4.1 GC-MS analysis of urine, plasma, and CSF, 269
10.4.2 LC-MS analysis of urine, blood, and CFS, 271
10.4.3 NMR analysis of CSF, urine, and blood, 274
10.5 Applications, 277
10.5.1 Identification and classification of metabolic disorders, 278
10.6 Future outlook, 283

INDEX 289
PREFACE

It has been less than a decade the word “metabolome” was first used referring to
all low molecular mass compounds synthesized and modified by a living cell or
organism. As a consequence, metabolomics emerged as a new field in the biologi-
cal science, achieving tremendous development and popularity in the last couple of
years. Many would say that metabolomics is a new word for an old science, because
it revives the classical biochemical concepts and studies what became “unfashion-
able” during the genomics era, and it makes extensive use of analytical techniques
idealized much earlier than the massive genome sequencing programmes. But, the
applicability of metabolomics combined with genomic information or other sys-
tem wide approaches make this field unique in modern science, both because of
its multidisciplinary requirement, where biologist, chemists, engineers, physicists,
mathematicians, and statisticians have to join forces to solve common problems;
or by its ambition in connecting the different levels of biological information at the
molecular level.
As a postgenomics tool, metabolomics is a young field in science but in an expo-
nential growth phase. There is already a peer reviewed journal in its second year of
publication, totally dedicated to publish works in the metabolomics field (Metabo-
lomics, Springer), an international Metabolomics Society that was formed in 2004
(www.metabolomicssociety.org), and six annual international conferences focused
entirely on metabolomics developments and studies (the International Conference on
Plant Metabolomics and the Scientific Meeting of the Metabolomics Society).
Despite of all the advances in the metabolomics area, there has been a lack of
a concise and basic literature focused on metabolome analysis, particularly an
introductory text that can be used as a general guide for a novice interested to start
exploring this new field or as a textbook for graduate and undergraduate students

xiii
xiv PREFACE

attending specialized courses. We, professionals with different scientific back-

grounds, therefore joint efforts to write this textbook, aiming to guide the reader
to the main steps involved in metabolite analysis, and covering different biological
materials (e.g., from plant and animal tissues to microbial and cell cultures, body
fluids, and extracellular media), as well as presenting and discussing the principles
of the most used methodologies for sample preparation, separation techniques, and
detection methods.
The reader will find the book divided into two parts: Part I presents and discusses
the concepts and methodology behind metabolite analysis. We first introduced the
metabolomics field and its new terminologies (Chapter 1), followed by a general
introduction to the diverse biochemical world of small molecules, where the basic
concepts of cell metabolism are presented and the differences between primary and
secondary metabolites as well as the dynamics of biochemical reactions and me-
tabolite turnover are discussed (Chapter 2). Then, progressively, the reader is taken
through the several steps of metabolome analysis, starting with reviewing the diver-
sity of techniques used for sampling and sample preparation (Chapter 3), followed
by a global overview of modern analytical methods used in the separation, detection,
and identification of metabolites (Chapter 4) and ending with Chapter 5 that is fully
dedicated to the most challenging aspect of metabolomics—the data analysis.
Part II of the book is aimed to illustrate the applicability of metabolomics and to
discuss specific particularities and requirements of metabolomics in certain groups
of organisms. Thereby, we review successful cases of metabolome analysis, illus-
trating yeast metabolomics (Chapter 6); reviewing specialized sampling devices for
microbial metabolomics (Chapter 7); discussing the plant systems and reviewing the
major achievements in plant metabolomics (Chapter 8); illustrating the applicability
of metabolomics in the classification of filamentous fungi (Chapter 9); and finish-
ing the book with a complete review of metabolomics applied to human and other
mammals (Chapter 10).
Our goal as authors was to write a concise and practical focused book as an
introduction to metabolome analysis. A book focused on an integrated analytical
approach combining the whole analytical chain from sampling over extraction and
separation to state-of-the art mass spectrometry and data processing. Although we
included a few review chapters in the second part of the book, it is important to
emphasize that this book was not intended to be a review book but a textbook that
introduces the principles rather than the latest results. The readers will find in the
next pages bits of biochemistry, bits of molecular biology, bits of analytical chemis-
try, bits of mathematics and statistics, and even bits of chemical engineering. That
was the challenges that we faced when decided to write this book: to organize the
work-flow in metabolome analysis covering all different biological systems and all
interdisciplinary aspect. We believe in metabolomics as a field per se rather than an
additional tool in science. We borrow tools from different sciences to build this new
field: METABOLOMICS. Now we invite you to try it.
LIST OF CONTRIBUTORS

Dr. David Wishart, Deptments of Biological Sciences & Computings Sciences,

2-21 Athabasca Hall, University of Alberta, Edmonton, AB Canada, T6G 2E8
Dr. Jens Nielsen, Center for Microbial Biotechnology, Building 223, BioCentrum-
DTU, Technical University of Denmark, DK-2800, Kongens Lyngby, Denmark
Dr. Jørn Smedsgaard, Center for Microbial Biotechnology, Building 221,
BioCentrum-DTU, Technical University of Denmark, DK-2800, Kongens Lyngby,
Denmark
Dr. Michael Adsetts Edberg Hansen, Center for Microbial Biotechnology, Build-
ing 223, BioCentrum-DTU, Technical University of Denmark, DK-2800, Kongens
Lyngby, Denmark
Dr. Silas Granato Villas-Bôas, AgResearch Limited, Grasslands Research Centre,
Tennent Drive, Private Bag 11008, Palmerston North, New Zealand
Dr. Ute Roessner, Australian Centre for Plant Functional Genomics, School of
Botany, the University of Melbourne, 3010 Victoria, Australia

xv
PART I

CONCEPTS AND METHODOLOGY

1
METABOLOMICS IN FUNCTIONAL
GENOMICS AND SYSTEMS BIOLOGY

BY JENS NIELSEN

This chapter gives a brief introduction to the field of metabolomics and puts this in
perspective of the current development in molecular biology, where genomics have
resulted in a move from a reductionistic analysis of biological systems (or even sub-
systems) to a systems (or global) view on the function of biological systems. Thus, the
chapter serves as an introduction to the textbook.

1.1 FROM GENOMIC SEQUENCING TO FUNCTIONAL GENOMICS

In 1992 the first nucleotide sequence of a complete chromosome was obtained, namely
the DNA sequence of chromosome III of the yeast Saccharomyces cerevisiae, and
around the same time efforts to sequence the human genome were initiated. In 1995
the first complete genome was sequenced, namely that of the pathogenic bacterium
Haemophilus influenzae, and in 1996 the complete genomic sequence of the yeast S.
cerevisiae was released. Since then there has followed genomic sequences of many
different organisms (Figure 1.1), and currently the number of sequences entered into
GenBank is doubled every 10 months. Genomic sequences provide the blueprint for
cellular function, and the complete set of genes within a genome basically defines a
functional space for the organism. However, in order to further define this functional
space it is necessary (1) to know the function of all the proteins and (2) to know the
relationship between which genes are expressed (or which proteins are present) at
different environmental conditions. Since the first complete genome was released,

Metabolome Analysis: An Introduction, by Silas G. Villas-Bôas, Ute Roessner,

Michael A. E. Hansen, Jorn Smedsgaard and Jens Nielsen
Copyright © 2007 John Wiley & Sons, Inc.

3
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