About the Authors

Camilla B Hill
Camilla B Hill graduated from the Free University, (Berlin, Germany) with
a MSc (Diploma) degree in Biology in 2008. To accomplish the laboratory
work for her thesis in plant molecular biology, she went to the Washington
State University in Pullman (Washington, USA) on a DAAD fellowship (German Exchange Academic Service). She moved to Melbourne (Australia) in
2010 to start a PhD at the ACPFG (Australian Centre for Plant Functional
Genomics) at the University of Melbourne, under the supervision of Prof
Antony Bacic and A/Prof Ute Roessner. She applied metabolomics as a
new tool to identify drought tolerance-related QTL in a wheat mapping
population, and also worked on a project regarding plant salinity tolerance
in collaboration with researchers from the University of Adelaide node of
the ACPFG. She continues to work a Postdoctoral Researcher at the School
of BioSciences in Melbourne, currently focused on the development of
a multidisciplinary functional genomics approach using a combination of
physiological, molecular and analytical techniques to identify novel salinity
tolerance mechanisms in barley.

Ute Roessner
Ute Roessner has obtained her PhD in Biochemistry at the Max Planck
Institute for Molecular Plant Physiology in Germany, where she developed
novel analytical methods to analyze metabolites in plants. Together with
the application of sophisticated data mining the field of metabolomics was
born and is today an important tool in biological sciences, systems biology
and biomarker discovery. In 2003, she moved to Australia where she
established advanced metabolomics platforms as part of the Australian
Centre for Plant Functional Genomics for which she leads the node at the
University of Melbourne. In addition, since 2007 Ute Roessner has been
involved in the setup of Metabolomics Australia (MA), a federal and state
government funded national metabolomics service facility and now leads
the MA node at The University of Melbourne. In 2013 she was awarded
a Future Fellowship from the Australian Research Council to investigate
the role of lipids in salinity adaptation and tolerance in barley.

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Chapter

4
Advances in
high-throughput
untargeted LCMS
analysis for plant
metabolomics

Experimental
considerations for
untargeted LCMS-based
metabolomics
61
Advances in data
acquisition

64

Advances in data
processing & analysis

64

Applications of the
untargeted LCMS
approach

66

Challenges of untargeted
LCMS-based
metabolomics
67

Camilla B Hill & Ute Roessner

Plants produce an extensive chemical diversity of metabolites, estimated to be between 100,000 and 200,000
compounds. LCMS provides a tool for dissecting this
immense plant biodiversity due to this techniques
capability of analyzing a broad range of metabolites
including secondary metabolites (e.g., alkaloids, benzoids, flavonoids, terpenes, isoprenes, glucosinolates and
phenylpropanoids) and highly polar and/or higher molecular weight molecules (oligosaccharides and lipids). LC
MS is one of the major untargeted analytical techniques
to determine global metabolite profiles, which aims at
the identification and relative quantitation of all peaks
in the chromatogram as ions that are initially defined
by retention time and molecular mass. This chapter outlines the common untargeted metabolomics approaches
based on LCMS, focusing on applications in plants and
including the latest software packages and bioinformatics
tools capable of processing untargeted LCMS datasets in
a high throughput manner. It also discusses some challenges faced by untargeted metabolomics, as well as
recent advancements to help address some of these challenges.

doi:10.4155/fseb2013.14.54
c 2015 Future Science Ltd


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Plant-derived small-molecule metabolites participate both in plant growth
and development (mostly by primary metabolites) and in defense against
harmful environmental (biotic and abiotic) factors (mainly by secondary
metabolites). An intriguing observation is that despite the highly conserved
nature of primary metabolism there is extensive chemical diversity of the
secondary metabolism among plants, estimated to give rise to between
100,000 and 200,000 compounds in the plant kingdom [1], far in excess of
other organisms.
Because of the diversity of compound classes there is neither a single sample
preparation approach nor a single detection technique that can be used to
measure the complete metabolite composition of a sample without introducing bias toward certain groups of metabolites [2]. With the advancements
made in analytical technologies, most notably in GC coupled to MS and LC
coupled to MS, metabolic fingerprinting focuses on the untargeted and
high-throughput analysis of hundreds to thousands of compounds in a single sample. Primary metabolites including carbohydrates, amino acids and
organic acids in plants are often measured by GC-MS after derivatization.
On the other hand, many complex secondary metabolites, including phenolics, alkaloids, glucosinolates and terpenoids, among others, are usually
detected using LCMS.
For profiling plant metabolites, two major approaches are commonly used.
The first approach is targeted metabolomics. Targeted metabolomics, often
known as targeted metabolite analysis, aims at the analysis of compounds
for which standards are available and only measures a subset of the metabolites in a plant sample at one time. Targeted analysis is usually undertaken in
a quantitative manner with specialized sample preparation and metabolite
extraction protocols for more accurate measurements of the metabolites
compared with untargeted metabolomics. This often allows for a better
understanding of the metabolic pathways in which the measured metabolites are involved. One recent example of such an approach is a study
that measured flavonoids produced by Arabidopsis thaliana wild-type and
flavonoid biosynthetic mutant lines [3]. In combination with transcriptomic
data, putative flavonoid pathway genes were identified. As the availability
of commercial standard compounds for plant secondary metabolites is still
very limited, this technique cannot be used to analyze unknown metabolites
or to survey global metabolic changes. This can potentially lead to limitations
in plant metabolomics experiments because important metabolic features
may be overlooked.
In contrast to targeted metabolomics which usually measures known
metabolites, untargeted metabolomics aims at measuring all of the ions,
from both known and unknown metabolites that can be detected within
a certain mass range. Analytical strategies for untargeted metabolomics

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Advances in high-throughput untargeted LCMS analysis for plant

metabolomics
include metabolic profiling, metabolic fingerprinting and metabolic footprinting, and these are selected according to the focus of the research or
the research question asked [4]. Global metabolite profiles of plant samples
using a combination of different instrumental platforms and analytical techniques can improve the chances of revealing the complete set of differences
in the metabolome, including unanticipated perturbations of metabolism,
and can potentially lead to the discovery of novel metabolites and metabolic
pathways [5].
Due to the plant kingdoms extensive metabolite diversity, the use of LC
MS is of particular importance for untargeted plant metabolomics, and this
technique represents an important tool among the vast array of analytical
techniques currently employed in metabolomics. Here, we describe the
progress made in the high-throughput analysis of the chemical compositions
of plants using LCMS analytical platforms in an untargeted manner. We
also evaluate the potential for wider use of this strategy by focusing on the
application of untargeted metabolomics approaches and their integration
with other high-throughput technologies. In the final section of this chapter,
we describe the current challenges and limitations of untargeted LCMS
analyses in plant metabolomics.

Experimental considerations for untargeted LCMS-based

metabolomics
Minimizing unwanted sources of analytical variations during the experiment
is very important for untargeted analyses since these variations could affect
data analysis or mislead biological interpretation. Examples of the analytical
variations include the chemical contaminants and the electronic noise of the
instrument. By contrast, biological variability results from genetic variation
within the samples. Preanalytical variability includes environmental variations arising from poor experimental design of the field or laboratory trial
(i.e., not all of the preanalytical variables have been controlled), or variation
arising from perturbations during sample collection and processing.
To estimate these variations, the preparation and analysis of sufficient technical and biological replicates are required. Additionally, recent publications
suggest the use of a linear mixed model analysis as implemented in software
packages such as ASReml/R [6], which is useful to account for variations in
the data due to batch effects, shifts in retention time and changes in ion
intensities, which can potentially undermine the identification and semiquantitation of all metabolites [7,8]. The use of quality controls is highly
recommended for ensuring reproducible data and to determine if measurement errors are arising from laboratory procedures (for more detail, see
Chapters 10 and 12 of this book). The quality control samples can either be

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Figure 4.1 Workflow for an untargeted metabolomics experiment.
Field and/or laboratory

Sample collection
Plant tissue harvest by
freezing in liquid nitrogen
Store samples at -80C until
extraction
Weigh samples and note
down exact weight

LC-MS instrument

Sample preparation

Separation (LC)

Samples homogenization
(e.g., using a mortar and
pestle or cryo mill)
Sample extraction (e.g.,
using methanol/water
solution)
Transfer aliquots into
HPLC vial inserts

Detection (MS)

Use of reversed phase

phase-based separation

Q-TOF, TOF, FT-ICR, or

orbitrap mass analyzers

Use of aqueous normal

phase-based separation

Use of positive and/or

negative mode ionization

Dual column or tandem LC

operation, simultaneous
analysis with two different
columns

Full scan acquisition

MS/MS fragmentation*,
MSn*

In silico
Data extraction
Background ion subtraction
Untargeted mass peak
extraction
Alignment of mass features
across the data set
Normalization (sample
weight, internal standards)

Identification*
Validation using MS/MS-based
fragmentation from standards
Library search against accurate
mass and retention time*
NMR-based structure
elucidation

Multi variate statistics

Correlation analyses
Students t-test, ANOVA
Supervised classification:
PLS, PLS-DA, OPLS-DA.
Unsupervised classification:
PCA, HCA

Data interpretation
Identification of important
mass features, biomarkers, etc.
Generation of metabolic
pathway maps
Biological interpretation of
metabolite variation in the
data set

*optional

Advances in metabolite extraction and separation methods.

a mixture of aliquots of different extracts or a mixture of purified standards,

and are used to determine the repeatability of the LCMS analysis.
Because each analysis provides only a single snapshot of the biochemical
state of that sample, experiments need to be specifically designed to study
biological variation. The number of biological replicates (sample size) is
usually determined using power calculations, which require the estimation
of population means, standard deviations and effect sizes. Methods have
been designed to estimate sample sizes for multidimensional data such
as gene expression data [9]. However, these methods require previously
acquired pilot data, and although highly recommended, it is often not fiscally
feasible to perform a separate experiment to determine the optimal number
of biological samples for each group in untargeted metabolomics. As a
general rule, at least three to six independent biological replicates should
be used (depending on the type of study), and all samples should be run
in a completely randomized order to limit batch effects. Ideally, all samples
should be analyzed on the same day and in the same batch.
Figure 4.1 illustrates the general workflow for an untargeted LCMS experiment and indicates significant recent technical advances that are further
described in the following sections.

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Advances in high-throughput untargeted LCMS analysis for plant

metabolomics
Sample preparation (which includes adjustments to the extraction solvent
composition and temperature) makes a vital contribution to defining the
array of metabolite classes covered (for more details, see Chapter 4 of
this book). Recent papers have been published on developing methods
for sample preparation, and it has been strongly recommended to analyze
samples in a pilot project before processing the full sample set [10].
Advances have been made in the development of more comprehensive
sample preparation procedures through the use of combinations of different solvents (water, methanol and/or chloroform) for simultaneous extraction of many different plant compound classes, in combination with other
extraction methods such as solid phase extraction (SPE) [11].
After the extraction, samples are separated using LC. Careful selection of
an appropriate LC separation method is then necessary according to the
objectives of the respective study. The two most commonly used LC stationary phases used in LCMS based metabolomics are reversed phase (RP)
and the so-called hydrophilic interaction chromatography (HILIC) phase. In
RP liquid chromatography, retention and separation of different molecules
mainly depends on their hydrophobic interactions with the stationary phase
(usually hydrocarbon chains) chemically bonded to a polar silanol surface.
However, highly polar or ionic metabolites (e.g., sugars, amino acids and
organic acids) are not retained by RPLC columns. To retain and separate
polar and hydrophilic compounds, a complementary LC technique as HILIC
where retention and separation is based on hydrophilic interactions of the
molecules with the polar stationary phase of a HILIC column, is often useful.
RP and HILIC provide essentially orthogonal separation, as metabolites that
are strongly retained by one type of column, are weakly or not at all retained
by the other. When each is used individually, a significant fraction of the
metabolome is eluted in the unbound fraction with significant loss of information. A rather new development is to incorporate RP and HILIC methods in
a single analytical run. Two dimensional liquid chromatography (2D-LC) techniques, which are based on the coupling of two columns with complementary chemistries, are well established in the proteomics field, although the
first separation is usually done off-line. For example, peptides are resolved
with 2D-LC configurations that employ a strong cation exchange column
coupled with a RP column, which exploits the diversity in ionic strength and
hydrophobicity of peptides to analyze a greater number of peptides in the
sample.
The extensive chemical diversity and complexity of plant metabolites suggests the need for further developments in this area (e.g., by using one to
two dimensions of separation to increase peak capacity). Currently there are
only few publications that described the use of 2D or tandem LC methods for

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untargeted metabolomics analyses in plants using online separation [12,13].
If done online, these approaches present some technical difficulties in combining different solvent elution conditions that are compatible with RP and
HILIC-based LC resolution [14-16]. However, if the solvents are compatible
and the analysis is done in an online and automated fashion, this reduces
the number of injections per sample required to analyze a larger number
of different compound classes simultaneously. This can be very helpful for
cases where sample is limited, and avoids errors due to having to prepare
two separate samples.

Advances in data acquisition

Continuous developments are underway in the metabolomics field to
improve data acquisition in order to achieve better data quality. Recent
advances made by all mass spectrometer vendors have resulted in the
development of faster and more sensitive instrumentation with a greater
dynamic range. These improved instruments allow the analysis of many
more compounds in a shorter time frame, as well as enabling the detection
of low-abundance compounds among the higher-abundance compounds
in the complex plant extracts. Since LCMS is amenable to a wide range
of different chemistries (as opposed to GC-MS which is limited to smaller
metabolites which are either volatile or can be chemically altered to become
volatile), the aim is to use that feature of LCMS to separate and detect as
many metabolites as possible in one single analysis.
Advances in data processing & analysis
After the acquisition of LCMS data, the datasets are processed in silico,
which, depending on the software package used, can include mass spectral
deconvolution, peak detection, peak alignment, adjusting for baseline shifts,
peak filtering by p-value and/or fold change, normalization and generation
of final data matrices. Recent developments of the data processing software
packages for unbiased mass peak extraction and alignment of LCMS data,
such as MetAlign, MetaboAnalyst, metaXCMS and MZmine offer possibilities
for more holistic untargeted metabolomics even across multiple datasets
(e.g., using metaXCMS), and have as their goal to gather information on as
many metabolites as possible in each dataset without prior identification
(Table 4.1). If samples have been run using two different columns (e.g., RP
and HILIC) and/or in two different ionization modes (positive and negative),
data matrices may need to be combined into a single data matrix for a
holistic interpretation and visualization of metabolite profiles. If an identical
metabolite is detected by more than one LC method, or in both positive and
negative ionization mode, the data with the higher signal to-noise ratio are
often selected for subsequent quantitative analysis [17].

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metabolomics
Table 4.1. Selected freely available software packages for processing untargeted
LCMS datasets.
Software

Publisher

URL

Reference

metAlign

Plant Research
International, The
Netherlands

[18]

[19]

MetaboAnalyst

University of Alberta,
Canada

[20]

[21]

MZmine/MZmine2

Developed by Matej
Oresic (VTT Technical
Research Centre,
Finland) and Mikko
Katajamaa (Turku
Centre for
Biotechnology,
Finland)

[22]

[23]

XCMS/XCMS2

Scripps Research
Institute, USA

[24]

[25]

metaXCMS

Scripps Research
Institute, USA

[26]

[27]

An example of the vendor-specific software packages for processing untargeted LCMS and GC-MS datasets is the MassHunter Workstation and Mass
Profiler Professional software (Agilent Technologies, CA, USA). Some features of this software package are:


Compatibility with both LC and GC, and independent of mass

spectrometer detector type
Automated local noise calculation and mass-specific baseline corrections
Automated mass feature alignment across large data sets
Includes multivariate analysis tools such as PCA, ANOVA, hierarchical
clustering and class prediction algorithms
Automated searches for significant compounds against public or in-house
generated databases.

A detailed workflow using MassHunter Workstation software for the untargeted comparison of hundreds of LCMS data files is available in [28].
Subsequently, multivariate statistics are applied to deal with the analysis of
multiple variables simultaneously, and to determine which ion features correlate with the study design. Unsupervised classification methods such as
principal components analysis (PCA), hierarchical cluster analysis (HCA) and

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self-organization mapping (SOM), and supervised approaches such as partial
least squares (PLS) regression and its latest extensions orthogonal projections to latent structures (OPLS) and OPLS-DA (discriminant analysis) are
used to identify class differences within a multivariate data matrix [29]. Correlation analyses based on Pearsons correlation coefficient or Spearmans
rank coefficient, and the more recently applied Gaussian graphical models
(GGMs), which are based on so-called partial correlation coefficients [30], are
utilized to infer relationships between metabolites and are often depicted
as metabolic correlation networks. Tools such as VANTED [31], Cytoscape [32]
and MapMan [33] can be further used to place identified metabolites onto
metabolic pathway maps to visualize metabolic profiles.
This approach generally involves searching accurate masses against publicly available mass spectral libraries such as KEGG (Kyoto Encyclopedia of
Genes and Genomes [34]), MassBank [35] and Metlin [36], or in-house created libraries, to generate a list of potential candidate metabolites. As this
approach uses accurate mass data to identify putative candidates, it relies
heavily on the completeness of the databases searched, which often does
not result in an unambiguous identification. Furthermore, it is time consuming to perform additional analyses to confirm putative identifications
(e.g., by using MS/MS spectra). Since this adds additional time to the end of
a study, structure elucidation is often only attempted for those molecules
that show statistical significance (for more details on LCMS applications for
identification of the metabolome, see Chapter 11).

Applications of the untargeted LCMS approach

Recent examples of untargeted approaches using LCMS-based instrument
platforms in plant research include:


66

Studies of plant biochemical composition (e.g., the development of a

species-specific and publicly available tomato fruit metabolome
database) [37], or comparison of the metabolite composition of individual
organs or tissues in strawberry [38];
Studies of environmental perturbations (e.g., abiotic [sulfur deficiency in
A. thaliana [39]] and biotic stress [fungal resistance in maize [40]] factors);
Detection of stress biomarkers (e.g., the discovery of wound biomarkers
in A. thaliana using machine learning classification algorithms) [41];
Plant functional genomics (integration of metabolomics with genomics)
studies (e.g., for metabolic quantitative trait locus analysis [42,43] and for
metabolomics-assisted gene annotation) [44];
Studies of the effects of genetic modification on metabolites (e.g., for risk
assessment of genetically modified tomato) [45].

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Advances in high-throughput untargeted LCMS analysis for plant

metabolomics
Challenges of untargeted LCMS-based metabolomics
One of the limitations of untargeted LCMS-based metabolomics is that a
researcher has to carefully select appropriate LC separation methods and
chromatographic conditions for comprehensive analyses covering a broad
range of metabolites with different chemical classes a process which can be
both time- and labor-consuming. A large number of variables can influence
the analysis outcome and with that the quality of the resulting data. These
variables include the type and the quality of the solvents, the type and
size of the columns, the solvent flow rate and gradient, and the ionization
techniques can all significantly affect the results of an untargeted LCMS
experiment.
Furthermore, most researchers would like to shorten the analytical run
time per sample to achieve high-throughput analysis. This becomes difficult
considering the complexity of the compounds in a crude biological extract,
especially for resolving the structural isomers of some metabolites that are
usually elute closely by LC. For best deconvolution and quantification in LC
MS, a certain degree of separation is required to obtain sufficient sampling
points across a peak. This limits how steep a solvent gradient can be for
a LC separation, which has an impact on how long it takes to analyze a
sample set. Differences in wait times before analysis raise issues of sample
stability and potential compound degradation. For very large experiments,
samples need to be separated into smaller batches to avoid these issues.
However, this may introduce other complexities such as data integration
across multiple batches. This may be overcome by introducing appropriate
quality-control samples such the pooled biological replicates recommended
by [46].
One of the major issues in untargeted metabolomics is metabolite identification of interesting features, which is often performed by searching publicly
available mass spectral databases and/or verification by comparison of the
retention times and MS/MS spectra of authentic standards, or by de novo
mass spectral interpretation. Due to both the limited accuracy of most nonFT mass spectrometers and the existence of structural or positional isomers
leading to isobaric species, one mass can have more than one putative
identification, and the required verification of the compounds identity by
measuring standard compounds can be costly and labor intensive.
Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any

organization or entity with a financial interest in or financial conflict with
the subject matter or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties.

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Hill & Roessner

No writing assistance was utilized in the production of this manuscript.
Summary


The plant kingdom shows extensive diversity of compound structures, physical and chemical
properties, and the wide concentration range of plant metabolites.
LCMS provides a tool to dissect this immense plant diversity due to its capability of analyzing a
broad range of metabolites including primary and secondary molecules.
Untargeted metabolomics has as its goal to measure all ions detected within a certain mass range
by MS, which may lead to the discovery of novel metabolites and metabolic pathways.
Care must be taken during data acquisition to avoid including ions coming from extraneous
sources.
Appropriate separation methods and chromatographic conditions for comprehensive analyses
covering a broad range of metabolites with different chemistries have to be carefully selected as
they can significantly impact the quality of data acquired in the experiment.
Recent developments in data processing software allow for automatic mass peak extraction and
alignment of LCMS data across multiple datasets, and these software packages are designed to
gather information on as many metabolites as possible in each extract.
New developments such as online 2D-LC techniques incorporate reversed phase and the
hydrophilic interaction chromatography methods in a single analytical run and can significantly
reduce sample numbers and preparation time.
Recent examples of untargeted approaches using LCMS-based instrument platforms in plant
research include studies on plant biochemical composition, environmental perturbations, as well
as studies to determine gene function by integration of metabolomics with genomics studies.

Key Terms
Metabolic profiling:

the determination and comparison of as many metabolites as

possible in a structurally related predefined group (e.g., organic
acids, amino acids and carbohydrates).

Metabolic fingerprinting:

in this technique, the full metabolite profile is treated as a

fingerprint or pattern of the metabolome within a biological sample
for comparison by nontargeted high-throughput analysis. This is
particularly useful in combination with pattern recognition and
discriminant analysis techniques.

Metabolic footprinting:

this technique is related to metabolic fingerprinting and involves

analyses of small molecule metabolites of microbial or cell cultures
secreted into the growth medium.

Targeted metabolite analysis:

this technique is used to determine the concentration of a limited

number of known metabolites, typically using multiple-reaction
monitoring on a triple-quadrupole mass spectrometer. Targeted
metabolite analysis provides very low limits of detection and
therefore lower limits of quantitation due to the high sensitivity and

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Advances in high-throughput untargeted LCMS analysis for plant

metabolomics
high specificity of multiple-reaction monitoring. The major
limitation is that it requires the standard compounds to measure
the absolute concentrations, and is usually only used for known
metabolites.

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