Imaging mass spectrometry of natural products

Eduardo Esquenazi,
ac
Yu-Liang Yang,
a
Jeramie Watrous,
ab
William H. Gerwick
ac
and Pieter C. Dorrestein
*
abc
Received 30th July 2009
First published as an Advance Article on the web 9th November 2009
DOI: 10.1039/b915674g
Covering: up to June 2009
This Highlight article describes three different imaging mass spectrometry (IMS) approaches,
MALDI, DESI and SIMS and their recent applications in the analysis of natural products. IMS
has opened up a new avenue for establishing the functional roles of natural products.
Introduction
Mass spectrometry (MS) has become an indispensable tool to the
modern natural product scientist by providing investigators with
molecular formulas, isotopic proles and fragmentation data
that are especially useful for dereplication and structure
elucidation. Molecules observed by mass spectrometry are
typically displayed as a plot of the mass to charge ratio (m/z)
versus ion intensity; however, it is also possible to add a spatial
dimension to the data by acquiring many mass spectra across the
surface of a sample in a predened sampling pattern, also
known as rastering. The intensity values of each ion can then be
highlighted across the sampled area to create an image showing
not only the spatial distribution, but also the relative intensity of
the ion throughout the sample. This is the basic principle behind
most forms of imaging mass spectrometry today.
While imaging mass spectrometry (IMS) has been around for
some time, with initial reports of the Secondary Ion Mass
Spectrometry (SIMS) imaging of solid surfaces as early as the
1960s by Raimond Castaing,
1
it has only in the past decade been
applied to biology due largely to the advancement of soft
ionization techniques. Commercial availability and renement of
the most common methods used for imaging biological
samplesMALDI (Matrix Assisted Laser Desorption Ioniza-
tion), SIMS (Secondary Ion Mass Spectrometry) and DESI
(Desorption Electrospray Ionization)as well as many other
forms of ionization, has allowed IMS to gain momentum in the
past few years.
213
While most of the imaging mass spectrometry
methodology developed in the past decade has been focused on
the discovery of biomarkers for disease, the tools developed for
this purpose have become important to our understanding of the
metabolism of therapeutic agents, which is rapidly becoming one
of the main applications of IMS.
In recent years, IMS has gained a wider utilization not only in
the characterization of metabolism of therapeutics, but also in
the understanding of their pharmacokinetic properties.
7,1416
Traditionally, such experiments are carried out by synthesizing
a radiolabeled drug, uorescent analog, or antibody followed by
visual analysis by autoradiography, uorescence imaging or
immunouorescence, respectively.
1722
Mass spectrometry,
including IMS, can be performed on any substrate provided that
the molecules ionize well enough and are present in sufcient
quantities to be detected. Additionally, imaging mass spec-
trometry not only reports on the location of the molecule, as is
the case for autoradiography or uorescence, but also provides
mass information on the molecules that are visualized and
therefore enables the visualization of the metabolic state of the
therapeutic agent. Even though small molecule imaging mass
spectrometry has become commonplace in pharmaceutical
companies for monitoring the uptake, bioavailability and
metabolism of therapeutics, there are only a few examples where
these techniques have been applied in natural products research.
In this Highlight article we want to emphasize the application of
IMS to the investigation of natural products, dened here as
metabolites that are directly observed from living organisms that
possess biological functions (e.g. polyketides, nonribosomal
peptides, alkaloids, lantibiotics, microcins, avinoids, terpenes
etc.). Our goal in this review is to introduce natural product
researchers to imaging mass spectrometry and highlight the
recent applications of natural product IMS that address the
functional roles of natural products in metabolic exchange,
including their role in communication, defense, and as entities
that control morphological changes in organisms and how
they may aid in the spatial analysis of natural products from
heterogeneous samples.
What is imaging mass spectrometry?
Imaging mass spectrometry, across all modalities to date, refers
to the idea of acquiring the spatial distribution of the chemical
composition of a sample. There are three main steps to the IMS
workow. The rst is sample preparation (Fig. 1A). Ionization
and desorption of molecules from the sample surface requires
varying amounts of preparation before analysis depending on the
technique used. MALDI utilizes a colloidal coating or organic
matrix to assist in the analyte ionization and desorption while
other techniques, including SIMS and DESI, require little to no
a
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of
California, San Diego, USA. E-mail: pdorrestein@ucsd.edu
b
Departments of Pharmacology, Chemistry and Biochemistry, University
of California, San Diego, USA
c
Center for Marine Biotechnology and Biomedicine, Scripps Institution of
Oceanography, University of California, San Diego, USA
These authors contributed equally to this work.
This journal is The Royal Society of Chemistry 2009 Nat. Prod. Rep., 2009, 26, 15211534 | 1521
HIGHLIGHT www.rsc.org/npr | Natural Product Reports
sample preparation before analysis. Reproducible sample prep-
aration, assisted desorption or not, is still one of the most chal-
lenging aspects for all IMS approaches at the present time and
therefore will be discussed throughout the text. The second step
in the IMS workow is data acquisition. In this process a user-
controlled stage containing the prepared sample is moved in
a dened geometry as the ionizer probe of the mass spectrometer
ionizes the area of interest to obtain data that is used for
generating an image. The term ionizer probe refers to the
method and mechanism of the ion source, and is responsible for
converting a liquid, solid or condensed phase sample into gas
phase ions detectable by the instrument. Currently, the most
common forms are a pulsed laser, a solvent spray or an ion beam
Eduardo Esquenazi
Eduardo Esquenazi was born in
Bogota Colombia in 1975 and
graduated from the Vanderbilt
University with a double major
in Biology and Neuroscience in
1998. He is pursuing graduate
studies at the University of
California, San Diego, depart-
ment of biology. As a joint
member of the Gerwick Lab at
the Scripps Institution of
Oceanography and the Dorres-
tein Lab at UCSD, Eduardo
utilizes novel mass spectrometry
approaches, including MALDI imaging, to reveal the spatial
features and temporal dynamics of marine natural product
biosynthesis.
Yu-Liang Yang
Yu-Liang Yang was born in
Hualien, Taiwan, in 1977. He
received his BSc in pharmacy
(1999) and PhD (2004) in
pharmaceutical science from
Kaohsiung Medical University
(KMU), Taiwan. At KMU,
under the guidance of Prof.
Yang-Chang Wu, he elucidated
the bioactive natural products
from Annona squamosa and
several herb medicines in Tai-
wan. From 2004 to 2008 he was
a Defense Industry Reserve
Duty Ofcer and postdoctoral research associate in the Institute of
Biological Chemistry, Academia Sinica, Taiwan (under Dr Shih-
Hsiung Wu), where his research focused on structures of bioactive
carbohydrates and secondary metabolites of thermophilic micro-
organisms. Currently he is a postdoctoral research associate in
Skaggs School of Pharmacy and Pharmaceutical Science,
University of California, San Diego, with Pieter C. Dorrestein
studying imaging mass spectrometry of natural products.
Jeramie Watrous
Jeramie Watrous received his
BS degree in biological chem-
istry from California State
University, San Bernardino in
2006. Following graduation he
began working in analytical
research and development at
Watson Pharmaceuticals where
he developed and validated
testing methods for upcoming
drug products. Currently as
a PhD student in biological
chemistry at the University of
California, San Diego, as
a member of the Dorrestein laboratory, Jeramie is continuing in
this area of research by working to improve and develop methods
for studying biological systems using mass spectrometry.
Bill Gerwick
Bill Gerwick received his BS in
Biochemistry at UC Davis
(1976) and PhD in Oceanog-
raphy at Scripps Institution of
Oceanography (Bill Fenical,
1981), did postdoctoral studies
at the University of Connecticut
(Steven Gould, 198182), was
professor of chemistry at the
University of Puerto Rico
(198284) and then moved
to the College of Pharmacy,
Oregon State University (1984
2005) as professor of pharma-
ceutical sciences. In 2005 he moved to his current position as
Professor of Oceanography and Pharmaceutical Sciences at
Scripps Institution of Oceanography and the Skaggs School of
Pharmacy, UCSD. His research focuses on discovery of novel
natural products from marine cyanobacteria, their biomedical
applications and biosynthesis.
Pieter Dorrestein
Pieter C. Dorrestein received his
BS at Northern Arizona
University (MacDonald, 1998)
and PhD from Cornell Univer-
sity (Begley, 2004). As a NIH
NRSA post-doc fellow Dorres-
tein used high-resolution mass
spectrometry methods to inter-
rogate the biosynthesis of ther-
apeutic agents under the
supervision and co-sponsorship
of Dr Kelleher and Dr Walsh. In
2006, Dorrestein joined UCSD.
His research aims to develop
new mass spectrometry approaches to detect and characterize
natural products as well as their biosynthesis. Dorrestein has
received several honors, including the Beckman young investigator
award, the V-Foundation scholar award, the PhRMA foundation
award, a Lilly young investigator award in analytical chemistry
and enjoys funding from the Hearst Foundation and National
Institute of Health.
1522 | Nat. Prod. Rep., 2009, 26, 15211534 This journal is The Royal Society of Chemistry 2009
Fig. 1 Overview of imaging mass spectrometry. (A) A schematic outline of the workow in a typical imaging mass spectrometry experiment. The
sample preparation can be assisted by a colloid or an organic matrix to aid the ion ionization or it can be unassisted and rely on the mechanism of the ion
source or the specic properties of the analytes that are ionized. Once prepared, sample data can be obtained in one of two approaches: spot raster
or continuous raster followed by time binning of the data to obtain a spectrum. (B) A schematic of the data processing. Once the spectra that will be
used to generate an image are obtained, the data processing starts. In short, data processing is accomplished by displaying ions of interest from
a traditional m/z vs. intensity plot as a false color image that is superimposed on a photograph of the analyzed sample. (C) Example results of an imaging
experiment and data processing. For this image, two marine cyanobacteria, Lyngbya majuscula 3L and JHB, were placed upon a MALDI-TOF
target plate and coated with HCCA/DHB matrix using an airbrush. The imaging was accomplished with a spot raster and the data processed using
FlexImaging 2.0. The ion corresponding to jamaicamide B (m/z 511, [M + Na]
+
) was colored in green and the curacin A (m/z 374, [M + H]
+
) colored
in red (A). The false color images were superimposed on a photograph of the marine cyanobacterial laments (B, C). Image C is adapted from
reference 38 with permission.
This journal is The Royal Society of Chemistry 2009 Nat. Prod. Rep., 2009, 26, 15211534 | 1523
gun; however, many other ion sources can be used for imaging.
2328
As the ions are emitted from the sample into the mass spec-
trometer, the mass analyzer (ion trap, TOF, ICR, ORBI) and
detector record the mass over charge ratios (m/z) of the analytes.
Depending on their concentrations in the sample, the ions have
different relative distributions as the ionizer probe rasters
a sample. Usually, mass spectrometry data is recorded as a graph
with m/z on the x-axis and relative ion intensity on the y-axis.
However, in IMS, an ion of interest is represented as a false color
image that is placed on top of a photograph of the sample that
was subjected to analysis (Fig. 1B). The more intense the ion in
the mass spectrum, the more intense the color is represented
in the 2D image (Fig. 1C).
At the present time there are two main approaches by which
the data for IMS is acquired. The approaches are spot rastering
or continuous rastering (Fig. 1A). Most previous studies have
relied on dened raster sampling spots. This is akin to a televi-
sion screen or monitor where different pixels, each containing
the color information for that particular spot, when viewed
together form an image. As is the case with pixels, the higher
the number of spots on a raster grid, the higher the resolution
of the image. In the case of imaging MS, the raster spots each
contain m/z data instead of color information. The spectral
signals from each coordinate or raster spot can then be
combined to yield the spatial distribution of the m/z values
across the sample. The continuous raster however, is an alter-
native to the raster spot approach and many IMS approaches
are based on this principle.
2931
In continuous rastering the
ionization probe scans across a sample at a constant speed
while recording mass spectra. The image is then generated by
time binning of the signals to display the image. The benet
here is that a continuous stream of data is gathered, and
resolution is only limited by the subsequent binning of the data
and the step size between each row; however, data may be lost
due to limited sample in a given region of the raster scan.
Therefore, the speed of the continuous raster needs to be
optimized to obtain the best quality signal. In the end, a single
IMS image is generated from the combination of hundreds to
thousands of individual mass spectra.
Practically, there are two components used to facilitate the
IMS process. First, there needs to be a user-controlled stage
that allows precise x and y movement. The quality of the user-
controlled stage can greatly enhance resolution, robustness and
repeatability of sampling patterns. In principle, any mass
spectrometer with an xy-stage can be used for IMS. Modern
MALDI-TOFs or MALDI ion sources, for example, all come
with this capability, and most other commercially available ion
sources that are used for imaging, such as DESI and SIMS, are
also now equipped with xy control. Second, a visualization
software package that allows specic m/z values to be given
a color and intensity and then co-localized to a light micro-
scopic image of the sample helps simplify data analysis and nal
image presentation. The end result of imaging by mass spec-
trometry is a snapshot of the density and distribution of
chemical substances contained in a sample, without the need for
labels, probes or pigments. This brings us to the third step of
the IMS workow: data analysis. There are several software
packages routinely in use for data analysis and visualization of
mass spectral data including the freeware MITICS and BioMap
(www.maldi-msi.org), as well as software by the manufacturers
of mass spectrometry equipment such as FlexImaging by
Bruker and ImageQuest by Thermo Scientic. Surprisingly,
even though IMS is close to becoming routine in life sciences
research, most mass spectrometry companies have not devel-
oped in house software to support this branch of mass
spectrometry. Rather, they rely on the analysis of IMS data
using freeware such as BioMap developed by Novartis and
others.
32
The availability of IMS freeware has been a huge asset
to the IMS community. Selecting the appropriate software
largely depends on compatibility, as there are issues arising
from the formatting of the data. There is an effort to stan-
dardize the le format for IMS, which if successful, would spur
new algorithm development and enable the routine use of these
tools in non-mass spectrometry laboratories.
This review article describes the use of imaging mass
spectrometry in natural product research applications. The three
main IMS approaches that have been used in natural
products investigations are MALDI-TOF, SIMS-TOF and
DESI-ion trap analysis. Even though they are all mass spec-
trometry approaches, each of these IMS techniques provides
different and complementary types of information that are
capable of solving diverse problems in biology. In the following
sections we describe the application of MALDI, SIMS and DESI
to the study of natural products through IMS.
Natural product MALDI-TOF imaging
MALDI time-of-ight (TOF) imaging is currently the most
widely accessible IMS technique. Because MALDI is commonly
interfaced with a TOF detector, which is the only detector used
in conjunction with MALDI to study natural products, this
instrument conguration is highlighted below. MALDI utilizes
a matrix to absorb laser energy which subsequently causes
desorption of the molecules to be detected in the mass spec-
trometer (Fig. 2A). MALDI was initially discovered in 1985
by Franz Hillencamp and Michael Karas who observed that
alanine could be more easily ionized in the presence of trypto-
phan. This matrix assisted principal was later applied to proteins
by Koichi Tanaka who was recognized with the Nobel Prize
in 2002 for his development of MALDI. Using the MALDI
approach for imaging was initially performed in 1994 by the
Kaufmann laboratory.
33
Since the initial applications of
MALDI-TOF to IMS, Caprioli, Sweedler, Salzet and many
other laboratories have expanded its use in biomarker discovery
and in the neurosciences.
3436
However, it should be noted that
MALDI-imaging has been accomplished using various detectors
other than TOFs, including ion traps, Orbitraps, Ion cyclotron
resonance (ICR) and other types of detector. With so many
possibilities, the eld of MALDI-TOF IMS is still young even
after a decade, and there are major ongoing research efforts to
improve sample preparation, instrumentation, resolution and
development of software.
3,5
To this end, MALDI-TOF IMS is of
growing utility in the natural products sciences by providing
spatial and temporal resolution of secondary metabolites from
marine sponges,
37,38
cyanobacteria,
37,38
zoanthids,
39
bacteria,
40
and plants.
4145
As mentioned above, in MALDI imaging a sample is covered
by a matrix that can absorb the energy emitted by the laser and
1524 | Nat. Prod. Rep., 2009, 26, 15211534 This journal is The Royal Society of Chemistry 2009
Fig. 2 MALDI imaging. (A) A schematic of laser desorption ionization (LDI) and matrix assisted laser desorption ionization (MALDI) to generate mass
spectrometry detectable ions. (B) Imaging mass spectrometry of a sponge demonstrating that different natural product microenvironments can be observed.
(a) Photograph of a cryosectioned Dysidea herbacea sponge with the region that was subjected to npMALDI-I highlighted. (b) Epiuorescence of the sponge
at 590 nm. (c) Epiuorescence of D. herbacea thin section at 420 nm. (d) MALDI image of m/z 530. This mass and the complex molecular ion isotope cluster
suggest its identity as the hexachlorinated peptide 13-demethylisodysidenin. (e) Autouorescence (420 nm) image overlaid by MALDI image at m/z 530
indicating localization of compound with this mass. (f) MALDI image of unknown compound m/z 1028. (g) Autouorescence (420 nm) image overlaid with
m/z 530 (red) and m/z 1028 (green) showing the differential localization of these two molecules. (C) Imaging reveals nonuniform glucosinolate distribution in
A. thaliana leaves. (a) Five different leaves showing observed 4MSOB ion patterns. (b) And (c) ion intensity maps of I3M (m/z 477 0.25) and 8MSOO
(m/z 492 0.25), respectively, obtained from mass data measured on the next to last leaf in (a). (d) A scatter plot of 4MSOB vs. I3M. The correspondence with
a line of slope y x shows the extent to which the two compounds co-occur. Scales are in false color intensity (0255) for both x and y axes. (e) A scatter plot
of 4MSOB vs. 8MSOO. Images B and C are adapted from references 38 and 42, Copyright (2009) National Academy of Sciences, U.S.A., with permission.
This journal is The Royal Society of Chemistry 2009 Nat. Prod. Rep., 2009, 26, 15211534 | 1525
transmit it to the molecules present in the sample (Fig. 2A). The
laser generates a plume of matrix and analyte ions which are
passed on to the mass detector. The sample preparation in the
MALDI imaging workow is by far the most important and
challenging step in the process. A sample (e.g. a thinly sectioned
sample or the organisms themselves if they are sufciently
thin) is prepared by mounting it onto a metal target plate or
conductive glass slide. The conductive plates are required to
enable the application of a voltage that accelerates the newly
formed ions towards the detector after desorption. Once the
sample is mounted a layer of a matrix is used to coat the surface
of the sample. Usually the matrix consists of small, light
absorbing, acidic molecules that act to softly, but efciently,
absorb and transfer the light energy from the laser into the
sample, yielding the ionization of a wide variety of molecular
species, including small molecules. While varieties of benzoic
acid, sinapinic acid, and cinnamic acid are the most common
components of MALDI matrices, there are many others, for
example conductive colloids such as graphite or gold, which can
be deposited on the sample in a homogenous layer. The appli-
cation of matrix to the sample surface can be performed by
many different methods such as spraying as a solution and
allowing it to dry, sublimation, applying with a ne sieve, or by
spotting.
4649
The choice of matrix and application method needs
to be optimized for each sample that is analyzed as different
matrices will ionize complementary sets of ions, especially when
switching from positive to negative ion mode. Depending on the
conditions and specic application, the matrix itself can act as
a proton donor or acceptor, thus helping to ionize the sample.
The variety of matrix molecules commonly in use is rapidly
expanding and is a very active area of research. With regards to
natural products research (although not applied to MALDI
imaging yet), a recent report by Shroff using a proton sponge
demonstrated that interference from matrix peaks can be
effectively eliminated, resulting in a clean spectrum in the lower
m/z region.
50
A number of recent reports provide excellent
coverage of the different varieties of MALDI matrices, their
preparation and function.
5154
Once the sample is covered by a matrix (or conductive
colloid), the prepared sample is ready to be placed on a user-
controlled stage under high-vacuum (10
6
torr) inside the
mass spectrometer. At this point, the area of the raster (spot
or continuous) is dened and subjected to IMS. The choice of
raster type is usually dependent on instrument capability, the
software interface that controls the instrument and the data
processing interface. The rastering is achieved by moving the
sample stage in an xy direction with respect to the laser.
When the laser hits the matrix covered sample, the material is
ablated, erupting into a plume of matrix and analyte mole-
cules. The molecules are then accelerated through the ion
optics and into the mass analyzer and detector. The result is
a complete m/z signal for that position on the MALDI plate.
As the sampling continues, either as raster spots or contin-
uous scanning, the MS data are stored along with the spatial
information. The data is then averaged as a whole (or time
binned and then averaged), forming a map of m/z values that
can be superimposed onto a light photomicrograph image
of the original sample, taken either before or after matrix
deposition.
38
To date, most studies of natural products using IMS have
employed MALDI-type approaches. IMS of natural products is
often referred to as natural product MALDI-imaging or
npMALDI-I to reect the types of interesting ions that are
being analyzed. In the next section we highlight the forefront of
npMALDI-I. At the present time, npMALDI-I has been limited
to marine cyanobacteria, marine sponges and plants. These few
examples demonstrate that IMS is able to provide the spatial
distribution of natural products in biological samples. In the
analysis of marine natural products, lamentous and non-la-
mentous cyanobacteria (e.g. Fig. 1C) and the marine sponge
Dysidea herbacea (Fig. 2B) are the principal organisms that
have been imaged by mass spectrometry for their natural
products. However, there is one report that used MALDI-TOF
proling to determine the spatial distribution of a marine
natural product, norzoanthamine in the zoanthid Zoanthus sp.
39
In these cases, MALDI imaging has provided us with a truly
unique look at classes of organism that have been consistently
shown to be the source of many powerfully bioactive, clinically
relevant, and commercially important secondary metabolites.
Indeed, the literature reveals that the range of bioactive
compounds isolated from marine organisms is stunning, with at
least 20 marine derived natural products currently or recently in
clinical trials for the treatment of cancer and many others
with promising activity in pain control, anti-microbial effects,
and anti-inammatory activity.
55
Many of these structures
and chemistries are novel when compared to their terrestrial
counterparts, probably stemming from their separate evolution
in a marine environment that has very different selection
pressures.
56
These proof-of-principle experiments provide
substantial support for the utility of imaging of marine natural
products by mass spectrometry.
One of the most intriguing applications of npMALDI-I lies in
the ability to provide spatial chemo-typing of complex assem-
blages of organisms, such as environmental samples of cyano-
bacteria, sponges, and many other marine invertebrates. The
literature has many examples where an organism that was
thought to produce a metabolite of importance was subsequently
determined to not be the true biosynthetic source. Symbiotic
microbes, cyanobacteria and ingested algae have been increas-
ingly found to be the true engines of the chemical diversity
present in nature.
57
In the case of marine sponges, genetic and
biochemical research in recent years has shown that symbionts
comprise nearly half the weight of the sponge and are responsible
for many of the described natural products.
5860
Thus, in essence
a sponge is really a miniature ecosystem comprised of a wide
variety of microbes and sponge cells which most likely interact in
much more complex ways than previously thought. By utilizing
MALDI imaging (using the spot raster method) the spatial
distribution of metabolites in a cross section of Dysidea herbacea,
a marine sponge collected in Papua New Guinea, revealed
a staggering number of different metabolites, some with the
ngerprint of halogenation, and in a variety of different distri-
butions (Fig. 2B). For example, the isotopic signature for
13-demethylisodysidenin, a known metabolite of Dysidea her-
bacea, was observed among the nearly 100 ionic species found,
including many halogenated ions, from a 14 mm thin cryosection
of the sponge (Fig. 2B).
38
This analysis enabled, in conjunction
with other methods such as epiuorescence, visualization of the
1526 | Nat. Prod. Rep., 2009, 26, 15211534 This journal is The Royal Society of Chemistry 2009
microenvironments that exist within this sponge.
61
As is also the
case with cyanobacteria, this information provides insight into
the biosynthetic origin of these metabolites which have often
been erroneously reported or been undescribed. Such informa-
tion can help guide the identication of other potentially
bioactive natural products as well as greatly facilitate efforts to
clone and study biosynthetic genes and their expressed proteins.
Terrestrial plants are traditional and extraordinarily rich
sources of natural products, several of which have given rise to
important clinical agents useful for treating infectious diseases,
cancer, heart disease, and others. It is therefore not surprising
that npMALDI-I has also been performed on plants.
4145
Most
reports of IMS on plants have been limited to pesticides,
carbohydrates, primary metabolites and higher molecular weight
peptides and proteins.
27,41,45,6264
However, a recent report
exemplies the role of MALDI imaging in helping unravel the
chemical ecology of natural products found within plants.
42
In
this study, the authors observe that the feeding pattern of the
cotton bollworm on Arabidopsis thaliana leaves is consistently
concentrated on the inner lamina, while the periphery and
mid-vein were avoided. Using npMALDI-I, it was shown that
three different glucosinolates, known to function as feeding
deterrents, were heterogeneously distributed along the leaves of
Arabidopsis thaliana, with the highest concentrations found in
the avoided areas (Fig. 2C). In further support of this conclusion,
it was shown that the glucosinolate concentration encountered
by the worm in these areas was sufcient to deter feeding. The
sample preparation for the MALDI imaging experiment in this
case was accomplished using a spray-coat with a 9-aminoacridine
matrix (for negative ion mode) and raster spot sampling to create
the image maps. The relative ease of observing these correlations
(e.g. without producing mutant strains unable to produce
glucosinolates) highlights the efcacy of IMS approaches to
investigate natural products and chemical ecological phenomena
in plants.
Thin layer agar natural product MALDI imaging of bacterial
cultures
Thin layer agar natural product MALDI-TOF imaging is
a specic method used for studying microorganisms grown in
culture (Fig. 3A). Albeit simple in concept, it is a very useful
tool to observe the production of natural products by
microorganisms grown under various culture conditions as well
as the interplay between competing species. In the thin layer
npMALDI-I, the top of the MALDI plate is coated with
culture agar, microorganisms are grown on top of the agar for
a variable period, and then the sample is covered with a matrix
and analyzed by MALDI. The most successful method for the
application of matrix to this agar surface has been the sieve
method. In this method, a ne sieve is used to sprinkle the
sample with a uniform layer of matrix. The sieve method is very
convenient, cost effective and easy to carry out. Thin layer agar
npMALDI-I provides critical insights into the spatiotemporal
productions and distribution of natural products, especially
those secreted by microorganisms into the surrounding media.
Using this approach both the natural product output and the
accompanying morphological change of a host microorganism
can be observed and correlated. In addition, this method offers
a great tool for studying complex intra- and inter-species
metabolic exchange phenomena and communication between
colonies of microorganisms.
In the co-culturing experiment of Bacillus subtilis 3610 and
Streptomyces coelicolor A3(2) shown in Fig. 3B-1, thin layer agar
npMALDI-I enabled the observation of several known natural
products including surfactin (m/z 1075), plipastatin (m/z 1545)
and a partially characterized polyglutamate (m/z 715) from
B. subtilis and prodiginine (m/z 392), calcium dependent antibi-
otic (CDA, m/z 1536) and the morphogen SapB (m/z 2027) from
S. coelicolor. However, many other ions were observed from
both species during the course of this experiment, some of which
are likely new and not yet characterized.
40
This approach
provides great insight into multi-species bacterial interactions,
including an anti-correlation between the production of surfac-
tin, a lipopeptide antibiotic produced by B. subtilis, and the
production of the morphogen SapB produced by S. coelicolor.
This anti-correlation was veried in an independent experiment
where puried surfactin was applied to the agar surface next to
S. coelicolor and resulted in inhibiting the production of SapB as
well as silencing the production of CDA, providing evidence that
natural products secreted by bacteria not only kill or alter their
morphology but they also silence the defense mechanisms of
neighboring organisms. To conrm this conclusion, B. subtilis
bacB/sfp double deletion was co-cultured with S. coelicolor to
provide a control data set. Sfp is responsible for the phospho-
pantetheinylation of the thiolation domains found on the non-
ribosomal peptide synthetases and polyketide synthases,
65
and
BacB is responsible for bacilysin production.
66
In this control,
the production of SapB was observed throughout the entire
colony. In addition, the amount of CDA production was
signicantly increased (Fig. 3B-1).
40
Using this agar-based IMS
approach, time-dependent correlations can also be made. For
example, in an effort to nd a candidate prodiginine inducer
secreted by B. subtilis, an sfp deletion mutant of B. subtilis was
applied to the agar 5 mm away from the S. coelicolor colony
(Fig. 2B-2). In this experiment, the production of polyglutamate
was always observed within the B. subtilis colony while the
unknown compound m/z 655 was secreted into the medium at
20 h under these conditions. When the m/z 655 ion contacts the
colony of S. coelicolor, it begins to generate the red pigment
prodiginine. Our hypothesis for this scenario is that the m/z 655
ion is secreted by B. subtilis and when it reaches the S. coelicolor
colony, it induces the biosynthesis of the prodiginines as well as
the other new ions observed under these conditions. Because m/z
655 has not yet been puried or characterized, as was possible for
surfactin, it has not yet been possible to directly establish that
this is the true inducer. These examples highlight the utility of
IMS for studying the complex chemical interplay of bacteria
grown in culture
The main challenge of thin layer agar npMALDI-I, as with
any other IMS approach, is still the sample preparation step.
Although the sieve method has made imaging results reproduc-
ible, the background signals, not only from matrices but also
from the enriched agar medium, are additional areas of concern.
This technical challenge could be at least in part surmounted by
establishing an ion reference for the agar medium which becomes
especially critical when one is interested in searching for the
unknown metabolites of microorganisms.
This journal is The Royal Society of Chemistry 2009 Nat. Prod. Rep., 2009, 26, 15211534 | 1527
Fig. 3 An overview of thin layer agar natural product MALDI imaging of microbial chemical communication. (A) A schematic representation of the
thin layer agar natural product MALDI imaging approach. Step 1, layer a thin lm of agar on top of the MALDI target plate and inoculate a culture of
the microorganism (fungus, cyanobacterium or bacterium). Step 2, allow the microorganisms to grow. Step 3, cover the cultures and agar surface with
matrix. Step 4, subject to npMALDI-I. Step 5. Average mass spectra and highlight ions with colors of interest. Step 6, display ions as false color image on
a photograph. (B) The structures and representative examples of metabolic exchange that can be captured by thin layer agar npMALDI-I. (1) An image
of metabolic exchange between B. subtilis (WT vs. bac-/sfp-) and S. coelicolor. (2) A distance time course of the candidate elicitor of prodiginine, m/z 655,
observed surrounding the B. subtilis colony.
1528 | Nat. Prod. Rep., 2009, 26, 15211534 This journal is The Royal Society of Chemistry 2009
Present challenges and limitations in npMALDI-imaging
Even though npMALDI-I is providing unprecedented insight
into the spatial localization of natural products, the method
itself, just as with any other mass spectrometry method, faces
signicant challenges in its application to natural product
research. Perhaps the most signicant issue impacting the use of
MALDI in natural product research is that the matrix itself is
composed of small molecules and associated clusters that are
similar in size to many natural products of interest (typically
below 1000 Da). In some cases, this can lead to considerable
difculty in determining whether a specic compound or set of
compounds is originating from the sample or from the combi-
nation of matrix and other sample preparation factors (e.g.
agar). In such cases tandem mass spectrometry, isotopic feeding
studies or other controls can be used to conrm the identities and
origins of ionized components. For example, if the molecule
contains halogen atoms, then the characteristic halogen signa-
ture will be present in the isotopic prole. Furthermore, the
imaging capability of MALDI provides a unique advantage in
determining the source of a signal by allowing direct visualiza-
tion of its location within the imaging area. Localization of
a signal to a specic area of the sample increases condence that
the signal is truly an expressed natural product. Finally, because
the matrix has to be crystalline to work effectively, reproduc-
ibility in imaging with MALDI-TOF is challenging and results in
only a modest number of images of sufcient quality for publi-
cation, one reason why most IMS papers contain only a few
images. As a result of this limitation, there are many devices and
matrix approaches that are under development; however, at the
present time, the sieve method and sublimation of the matrix
onto the sample are providing the best reproducibility for
npMALDI-I. In our experience, the sieve method for matrix
application in conjunction with the thin layer agar npMALDI-I
approach for imaging microorganisms grown in culture is
successful 8090% of the time, but has yet to be successful
applied to the imaging of cyanobacterial laments.
One additional limitation of MALDI imaging compared to
other imaging modalities is its spatial resolution. For most
MALDI instruments sold today, the resolution is limited by the
laser beam width as well as the size of the crystals used in the
matrix deposition. The effect of both parameters typically yields
a resolution between 20200 mm. However, these are active areas
of research and are already improving, with the next generation
of instruments and sampling approaches achieving resolutions
below 20 mm; there is even one report of a resolution of less than
1 mm.
67
However, the main downfall of improved resolution is
a corresponding decrease in sensitivity, a natural consequence of
the area being sampled. For example, a space of 1 mm
2
has about
10
6
molecules, which begins to approach the sensitivity limit of
current mass spectrometers. Most current matrices introduce
additional disadvantages such as signal interference, signal
suppression and impact on resolution. To solve this problem, the
mass spectrometry community is developing a variety of non-
matrix ionization approaches as well as new lasers.
54,68,69
However, these new developments have yet to be applied to the
ionization or imaging of natural products. Without doubt, some
of these matrix-free laser desorption approaches will advance the
eld of npMALDI-I over the next few years.
Natural product DESI-imaging
Desorption electrospray ionization (DESI) imaging is a recently
developed soft ionization technique that is similar to electrospray
ionization (ESI) in principle. Briey, a charged solvent spray,
aimed directly at a specic area of the sample surface, is emitted
from a small nozzle or capillary and upon impact ionizes and
dislodges the analyte from the sample surface; subsequently, the
ionized analyte is directed into the atmospheric inlet of the mass
spectrometer (Fig. 4A). The exact mechanism by which ions are
produced is still under investigation, but appears to depend on
the phase of the charged solvent spray upon contact with the
sample surface.
70
Collision of larger droplets of spray solvent to
a wetted sample surface tend to favor a momentum-transfer
mechanism whereby analyte-containing droplets are desorbed
from the surface. Smaller droplets, which are nearly in a gaseous
phase, are thought to ionize the analyte via chemical sputtering,
possible in combination with momentum-transfer.
71
Neverthe-
less, the ionization process occurs under ambient conditions and
sample preparation is minimal, making this approach very
attractive for the imaging of native surfaces. In 2004, Cooks and
Fig. 4 (A) A schematic representation of DESI ionization. (B) DESI
mass spectra of C. serratus surface, showing that bromophycolides occur
on algal surfaces only in association with light-colored patches. Top:
DESI-MS image of bromophycolide A/B m/z 701 [M + Cl]

on C. ser-
ratus surface, indicating that bromophycolide hot spots correspond to
pale patches. Bottom: a representative mass spectrum from patch-free
algal surface before and after mechanical damage. Image B is adapted
from reference 74 with permission.
This journal is The Royal Society of Chemistry 2009 Nat. Prod. Rep., 2009, 26, 15211534 | 1529
colleagues initially showed that this approach could be used to
detect chemical warfare agents, explosives, and over-the counter
drugs from different surfaces such as plastics, leather, and human
skin.
72
Additionally, it was shown that DESI could measure
varying quantities of a metabolite in different locations of
a cross-section of a plant stem, thus setting the stage for DESI
imaging.
70,73
DESI imaging, as is the case for MALDI imaging, is
accomplished by moving a sample stage in a predened xy
direction under the DESI ionization source. To date, DESI
imaging has been performed only using continuous rastering
with the sample stage moving at a constant velocity and the
resolution of the image determined by the step size between
sampling rows. At each raster row mass spectra are continuously
acquired and once the entire grid has been sampled, the
spectra corresponding to the ions in question can be displayed in
color and superimposed onto an image of the sample. The
greater the abundance of a particular ion species at a given
position of the raster, the higher the intensity of color displayed,
allowing the distribution and abundance of ions on a surface to
be visualized.
15
To date, there are only a few reports on natural product DESI
imaging. However, a pair of parallel reports have recently
appeared which conrm that DESI imaging can be employed in
natural product research to yield novel and insightful results.
74,75
Using a combination of DESI approaches, it was shown that the
amount and distribution of a family of antifungal metabolites,
the bromophycolides, across the surface of the Fijian algae
Callophycus serratus served as a surface-mediated defense against
the pathogen Lindra thalassiae. Not only were the authors able to
detect the presence of these metabolites in the surface matrix of
the alga, but they were also able to show that these compounds
were located on white colored patches that cover about 5% of the
surface of the algae (Fig. 4B). Moreover, DESI was able to show
that these antifungal compounds increased upon damaging of
the surface of the algae (Fig. 4B) Furthermore, the concentra-
tions in these patches and damaged surfaces were present at
effective antifungal levels, providing support for the hypothesis
that the bromophycolides serve as defensive entities against
fungal invasions. This study exemplies the role that IMS can
play in helping to elucidate the native ecological role of natural
products.
Present challenges and limitations in DESI-imaging
DESI, unlike MALDI, is a rather new ionization method, and
therefore its development is not as advanced, especially in the
area of imaging. Obtaining an optimal analyte signal using DESI
is highly dependent on a number of geometrical and instrumental
parameters, thus creating technical challenges in its application.
These parameters vary greatly depending on the type of analyte
being analyzed (i.e. proteins versus therapeutic drugs) and
include the spray angle, spray solvent, spray voltage, solvent ow
rate, surface type, the distance and orientation of the spray
nebulizer relative to the sample surface, the gas pressure that is
applied to the nebulizer and the type of surface being analyzed.
71
An additional challenge in DESI is the inefcient desorption of
the analyte thus resulting in a lower signal and sensitivity. The
best images that have been obtained with DESI are of small
molecules, such as the many therapeutics used in the clinic today,
that carry a negative charge, that are easily protonated or that
carry a permanent positive charge. DESI-images of larger mole-
cules such as peptides or proteins are more limited, reecting the
current technical challenges in the detection of these ions.
However, the development of DESI andits applicationtoimaging
is important as it provides a complementary data set compared to
other ionization methods such as MALDI. DESI provides
information on the low m/z ions that are difcult to observe by
MALDI, and provides a method which allows the analysis of
surface chemistry under ambient conditions. MALDI, on the
other hand, provides a method for probing more deeply into
the subsurface chemistries of samples. As has been the case for
MALDI over the past 30 years, it is likely that DESI, either in
its current conguration or as a hybrid method, will become
a robust method for analyzing surface chemistries.
Natural product SIMS imaging
Secondary ion mass spectrometry (SIMS) is similar to DESI in
that very little sample preparation is necessary, but the ioniza-
tion, as it is in MALDI, occurs under high vacuum. The
basic principle behind SIMS was rst demonstrated in 1910 by
J. J. Thompson and subsequently independently developed as an
instrument in the 1960s by two groups, Liebl and Herzog
76
and
Raimond Castaing.
1
It has been widely used to interrogate the
composition of surfaces and thin lms and thus has been a key
player in material science and geophysics. SIMS is used in
various other elds and biomedical applications as well, such as
for clinical investigations. SIMS works by directing a focused ion
beam on the surface of the sample. The beam dislodges and emits
secondary charged ions from the surface layer that are then
directed to a mass spectrometer, most often a time-of-ight
instrument (Fig. 5). Many types of ion beams have been utilized
in SIMS, including primary types such as Cs
+
, Au
+
, Xe
+
and
others. These are best for probing atomic composition, especially
for very small molecules of less than 300 Da, as the ion beam
tends to fragment molecules as they are ionized. More recently,
the use of C
60
+
and Au
+
primary ion beams has allowed the
detection of larger molecules. In some cases even small proteins
can be accessed by SIMS, and this has begun the use of SIMS for
biological imaging. Much of the modern development and
implementation of SIMS imaging of biological surfaces has been
carried out by the laboratories of Vaidyanathan, Vickerman,
Winograd and Sweedler among others.
13
Like DESI and
MALDI imaging, SIMS imaging also hinges on a movable
sample stage and user-dened raster grid to indicate the general
area of interest and the number and location of spots to be
sampled. The main advantage of SIMS is resolution; the focused
ion beam is extremely narrow, thus allowing the raster spots to be
tightly arranged and resulting in resolution in the order of
nanometers.
In three pioneering reports by the Vaidyanathan and Brunelle
laboratories the utility of SIMS-IMS to natural products
research was demonstrated.
7779
These authors used a TOF-
SIMS equipped with a C
60
+
primary ion gun to analyze the lipo-
peptide antibiotic surfactin from an imprint of a B. subtilis
colony.
77
A silicon wafer was pressed upon a B. subtilis colony
and then quantitative SIMS-imaging was performed which
demonstrated that surfactin was present in higher quantities in
1530 | Nat. Prod. Rep., 2009, 26, 15211534 This journal is The Royal Society of Chemistry 2009
the center and edges of the bacterial colonies (Fig. 5B). Although
the SIMS image of the colony, accomplished with a Bi
3
+
cluster,
has far superior resolution (2 mm) enabling a much more precise
location of surfactins, a similar result was obtained with
B. subtilis using the thin layer agar npMALDI-I approach
described above, even though the resolution in this latter
experiment was at least 2550 fold less than that obtained by
SIMS.
40
Moreover, while the MALDI result was able to visualize
multiple molecules such as the plipastatins, polyglutamates and
subtilosins, it was not reported if such ions could be observed
using SIMS imaging. The SIMS advantage of higher spatial
resolution was highlighted in the aforementioned natural
product IMS reports by the Vaidyanathan laboratory.
78,79
The
presence, absence and differential distribution of the blue
pigmented actinorhodin and the red pigmented prodiginine
antibiotics were observed from cells obtained from a liquid
culture and from colonies of the streptomycete bacterium
S. coelicolor A3(2) (Fig. 5C). Noteworthy among their ndings
was the observation that liquid culture preparations, which are
most prevalent in research laboratories, contained both the red
and blue pigmented antibiotics. However, solid cultures which
more closely resemble natural soil populations of the bacterium,
contained a signicant signal only for the red pigments with
very little evidence of any produced actinorhodin. Thin layer
agar npMALDI-I of S. coelicolor supports this observation.
40
However, the high spatial resolution of SIMS made it possible to
detect amino-acid fragment ions in the extracellular space
between cells, suggesting the secretion of peptides or proteins into
the mediumby the culture. Obtaining data of this resolution is not
currently possible with npMALDI-I or any other IMS approach.
This report, using minimally prepared samples, was the rst
example of a direct visualization of secondary metabolites in
proximity to their site of production, thus highlighting the value
of this complementary IMS approach. In general, the greatly
improved resolution of SIMS enables a quality of IMS that
cannot be obtained with any of the other current IMS techniques.
Present challenges and limitations in SIMS-imaging
The main challenge of SIMS-imaging is that the analysis requires
a at surface. Biological samples are not usually at, and thus,
this requirement presents a limitation for routine analyses by
SIMS. The silicon wafer copy of the sample is a creative solution
as it prints the natural products onto a at surface; however,
this method is unlikely to be universally applicable to all systems
and molecules. Thus far using the imprinting technique, only one
natural product, surfactin, has been observed and other natural
products known to be produced by B. subtilis were not reported.
It is likely that this approach selectively enriches for certain
molecules and perhaps if one screens different surfaces with
different adsorption properties additional molecules may be
detected. The silicon wafer approach will undoubtedly become
useful to investigate subclasses of natural products in biology.
The other main limitation that has plagued SIMS for decades is
the inability to control excessive fragmentation. Until recently,
SIMS was essentially limited to ions smaller than 300 Da.
However, with recent advances using C
60
+
and Au
+
ion beams, it
has been possible to observe intact biological molecules.
Nevertheless, this remains one of the main challenges in applying
SIMS to a broader range of chemical and biological questions.
Should these limitations be overcome, the superior resolution of
SIMS could provide great insight into biological phenomena.
Already, observations of smaller ions such as amino acids
detected between S. coelicolor cells
79
or an ammonia gradient
observed between heterocysts of lamentous cyanobacteria, are
demonstrating the potential utility of this IMS technique.
80
Comparison of IMS approaches that have been applied
to the characterization of natural products
This review has highlighted the strengths and limitations of each
of the three current IMS methods that have been applied to
natural products research. As mentioned in the introduction,
these IMS methods, SIMS, DESI and MALDI, are truly
complementary as each provides different information that
when combined gives a more complete view of the state of
a biological system. Of these, MALDI is the most readily
Fig. 5 (A) A schematic representation of SIMS ionization. (B) Micro-
scope and TOF-SIMS image of surfactin from part of the swarming
pattern of the bacterium B. subtilis (168 sfp
+
) transferred onto a silicon
wafer. The color scale on the right indicates the relative amounts of
surfactin ions detected. (C) Scanning electron micrograph (SEM)
image (left) and SIMS image (right) of S. coelicolor grown in salt
stressed conditions showing the location of prodiginine natural products
(m/z 390395). Bar indicates 100 mm. Images B and C are adapted from
references 77 and 79 with permission.
This journal is The Royal Society of Chemistry 2009 Nat. Prod. Rep., 2009, 26, 15211534 | 1531
accessible and probably the most robust approach at the present
time, and therefore, will likely continue to dominate natural
product imaging. MALDI-imaging is so robust that even
non-specialists of mass spectrometry are beginning to use this
methodology, usually through local mass spectrometry core
facilities. Additionally, a wider utilization of MALDI has been
facilitated by easily used software and established methods in
the literature. DESI and SIMS are not as readily available in
most mass spectrometry cores and are thus usually limited to
laboratories that build or develop mass spectrometry tools.
Moreover, the signicant degree of optimization necessary to
analyze each new sample by DESI or SIMS imaging relegates
these techniques to laboratories of MS specialists. If a non-
specialist desires to use these techniques, it is usually necessary
to collaborate with a mass spectrometry group capable of
performing the analysis. Each of the three IMS approaches
discussed here has distinct advantages, some of which are
tabulated in Table 1. MALDI tolerates less than perfectly
smooth surfaces, is a subsurface technique capable of pene-
trating about 1525 mm into the sample, and it can ionize many
different molecular ions at the same time. It is also a more
sensitive method resulting in the largest dynamic range
compared to the other IMS approaches. On the other hand,
DESI provides information on molecules that are present on
surfaces and works under ambient conditions while SIMS has
the best resolution.
The future of imaging mass spectrometry approaches
to characterize natural products
Each of the natural product IMS reports described in this
Highlight article provides a glimpse of what the future of IMS
holds. Because more than 60% of all therapeutics in use today
derive in some fashion from natural products, these techniques
will be of importance to diverse areas of biomedical research.
82,83
Many of the IMS images that have been generated to date are
proof-of-principle of their applicability to natural products
research. In several cases, IMS has provided deeper insight into
the known chemical and ecological roles that natural products
are thought to play. However, from a natural products stand-
point, the current early developmental stage of IMS can be
compared to the earliest advances in NMR spectroscopy; we are
presently using the equivalent of a 60 MHz NMR. Without
doubt, over the next few decades we can look forward to
signicant advances in IMS approaches, in part mediated by
improved sample preparation, improved algorithms, improved
Table 1 Comparison of IMS approaches that have been applied to the characterization of natural products
Ion source Sample preparation Analyzers
a
Sensitivity/
resolution Limitation
Natural products observed
with the different IMS
approaches
b
MALDI Sample prep. is critical and
an art. The choices of
matrix and the methods
for covering samples with
matrix are trial and error.
TOF, ion trap, LTQ,
Q-TOF, FT-ICR,
Orbitrap
fmolzmol/
10100 mm
Matrix signals may
interfere with the low
m/z region; sample is
under high vacuum.
Nonribosomal Peptides: 13-
demethylisodysidenin,
38
CDA, surfactins,
plipastatins,
40
yanucamide.
37
Hybrid Peptides/
Polyketides: curacin
A,
37,38
jamaicamide A
and B,
37,38
prodiginine,
40
viridamides.
37
Flavonoids: kaempferol,
43
quercetin,
43
quercetin-3-
O-rhamnoside
43
Alkaloids:
Norzoanthamine
39
Lantibiotics: the
morphogen SapB,
40
subtilosin
40
DESI Sample prep. is minimal
and works under ambient
conditions. Instrument
parameters such as spray
angle, spray pressure and
relative geometries need
to be optimized for each
sample.
TOF, ion trap, LTQ,
QTOF, FT-ICR,
Orbitrap
fmolpmol/
40400 mm
At the present time limited
surfaces can be analyzed.
For example DESI
imaging on solid bacterial
cultures has not been
achieved. DESI has an
analyte washing effect.
Polyketides:
bromophycolide A/B,
74
bromophycolide E
74
SIMS Minimal prep. but sample
surface needs to be
especially clean and at.
TOF, TRIFT Varies with m/z/
100 nm100 mm
Low sensitivity for high
m/z (>1000) ions due
to fragmentation; C
60
+
,
Au
+
secondary ions has
improved this limitation.
Sample is under high
vacuum.
Nonribosomal Peptides:
surfactin
77
Hybrid Peptide/polyketide:
prodiginine
78,79
Polyketide: actinorhodin
79
Terpenes: hinokione,
81
hinokiol
81
Lignanoids: hinokinin,
81
hinokiresinol
81
a
Analyzers in bold have been applied to imaging of natural products.
b
This list is limited to natural products detected by imaging.
1532 | Nat. Prod. Rep., 2009, 26, 15211534 This journal is The Royal Society of Chemistry 2009
mass spectrometers, improved dynamic range and the develop-
ment of new ion sources that can be used for imaging.
One of the new approaches currently in the pipeline of IMS
development is the ion mobility MALDI imaging mass spec-
trometer.
84
This instrument has a T-wave ion guide that can
separate different classes of ions by drift time, or the ease with
which an ion is passed through a gas lled chamber while riding
on a wave of current. The addition of the mobility dimension can
help separate matrix associated molecules from analytes arising
from the sample. Similarly, molecules with different hydro-
dynamic radii can also be separated under these conditions. In
the subsequent data analysis, this added dimension can be visu-
alized in the data resulting in trendlines based on mobility as well
as mass, charge and intensity. Peptides, fatty acids, and sugars all
exhibit different trendlines.
84,85
For example, a crude extract
from Lyngbya majuscula readily separated halogenated
compounds such as jamaicamide, hectachlorin and barbamide
from all other ions (Dorrestein, Gerwick labs unpublished data).
The additional degree of separation provided by improving ion
mobility techniques promises that the spectra resulting from
a very complex mixture will be more easily analyzeda boon for
dereplication and novel compound detection.
Lastly, all natural product IMS experiments to date have been
conducted on low resolution ion traps or TOF mass analyzers;
however, the adaptation of high-resolution mass spectrometers
to IMS will signicantly enhance the quality and utility of the
data that can be obtained. Instruments employing high resolu-
tion Q-TOF, ICR and Orbitraps equipped with IMS capabilities
have been designed or are in the development stage, and are
becoming commercially available. Most of these instruments not
only have better mass accuracy but also have the ability to ef-
ciently induce fragmentation of the ions that are under investi-
gation.
8689
Because identication of the ions that are analyzed is
a major problem in all IMS approaches studied to date, it is likely
that fragmentation patterns in conjunction with smart algo-
rithmic approaches will enable improved compound determina-
tion. In turn, this will allow for rapid dereplication of known
compounds, structural classication and, in some cases, struc-
tural elucidation by mass spectrometry alone.
90,91,92
As the limit
of detection and dynamic range of mass spectrometers improve,
the quality and the richness of information will similarly
advance. Therefore, IMS is sure to become a routine tool in the
natural product scientists arsenal to aid in the discovery of new
molecular entities and their natural functions. In short, the
increasing value and quality of IMS will place it at the forefront
of natural products investigations in the decades to come. The
ability to spatially observe and record the quantity of various
natural products within their biological context is invaluable to
decoding the chemical interactions between as well as within
species, and this is ultimately at the core of understanding the
innate function of natural products.
Acknowledgements
Funding for the Dorrestein laboratory was provided by the
Beckman Foundation, the V-foundation for Cancer Research,
the Hearst Foundation, and NIGMS grant R01GM086283.
Support to the Gerwick lab was provided by NIH NS053398 and
CA52955. E.E. was supported by the NIH marine biotechnology
training grant GM067550 and J.W. was supported by the NIH
molecular biophysics training grant GM08326.
References
1 R. Castaing and G. Slodzian, Comptes Rendus Hebdomadaires Des
Seances De LAcademie Des Sciences, 1962, 255, 1893.
2 R. M. A. Heeren, D. F. Smith, J. Stauber, B. Kukrer-Kaletas and
L. MacAleese, J. Am. Soc. Mass Spectrom., 2009, 20, 1006.
3 T. A. Zimmerman, E. B. Monroe, K. R. Tucker, S. S. Rubakhin and
J. V. Sweedler, Methods Cell Biol., 2008, 89, 361.
4 L. A. McDonnell and R. M. A. Heeren, Mass Spectrom. Rev., 2007,
26, 606.
5 S. Francese, F. R. Dani, P. Traldi, G. Mastrobuoni, G. Pieraccini and
G. Moneti, Comb. Chem. High Throughput Screening, 2009, 12, 156.
6 E. H. Seeley and R. M. Caprioli, Proc. Natl. Acad. Sci. U. S. A., 2008,
105, 18126.
7 E. H. Seeley and R. M. Caprioli, Proteomics: Clin. Appl., 2008, 2,
1435.
8 K. E. Burnum, S. L. Frappier and R. M. Caprioli, Annu. Rev. Anal.
Chem., 2008, 1, 689.
9 D. S. Cornett, M. L. Reyzer, P. Chaurand and R. M. Caprioli, Nat.
Methods, 2007, 4, 828.
10 M. L. Reyzer and R. M. Caprioli, Curr. Opin. Chem. Biol., 2007, 11,
29.
11 J. M. Wiseman, D. R. Ifa, A. Venter and R. G. Cooks, Nat. Protoc.,
2008, 3, 517.
12 J. M. Wiseman, D. R. Ifa, Q. Y. Song and R. G. Cooks, Angew.
Chem., Int. Ed., 2006, 45, 7188.
13 S. G. Boxer, M. L. Kraft and P. K. Weber, Annu. Rev. Biophys., 2009,
38, 53.
14 V. Kertesz, G. J. Van Berkel, M. Vavrek, K. A. Koeplinger,
B. B. Schneider and T. R. Covey, Anal. Chem., 2008, 80, 5168.
15 J. M. Wiseman, D. R. Ifa, Y. X. Zhu, C. B. Kissinger, N. E. Manicke,
P. T. Kissinger and R. G. Cooks, Proc. Natl. Acad. Sci. U. S. A., 2008,
105, 18120.
16 Y. Sugiura and M. Setou, J. Neuroimmune Pharmacol., 2009, DOI:
10.1007/s11481-009-9162-6.
17 W. A. Weber, J. Czernin, M. E. Phelps and H. R. Herschman, Nat.
Clin. Pract. Oncol., 2008, 5, 44.
18 A. M. Neubauer, H. Sim, P. M. Winter, S. D. Caruthers,
T. A. Williams, J. D. Robertson, D. Sept, G. M. Lanza and
S. A. Wickline, Magn. Reson. Med., 2008, 60, 1353.
19 C. Sun, J. S. H. Lee and M. Q. Zhang, Adv. Drug Delivery Rev., 2008,
60, 1252.
20 S. Lee, K. Park, K. Kim, K. Choi and I. C. Kwon, Chem. Commun.,
2008, 4250.
21 D. G. Reid and P. S. Murphy, Drug Discovery Today, 2008, 13, 473.
22 A. J. Fischman, N. M. Alpert and R. H. Rubin, Clinical
Pharmacokinetics, 2002, 41, 581.
23 T. J. Garrett, M. C. Prieto-Conaway, V. Kovtoun, H. Bui,
N. Izgarian, G. Stafford and R. A. Yost, Int. J. Mass Spectrom.,
2007, 260, 166.
24 P. Nemes and A. Vertes, Anal. Chem., 2007, 79, 8098.
25 P. Nemes, A. A. Barton, Y. Li and A. Vertes, Anal. Chem., 2008, 80,
4575.
26 Y. Li, B. Shrestha and A. Vertes, Anal. Chem., 2007, 79, 523.
27 Y. Li, B. Shrestha and A. Vertes, Anal. Chem., 2008, 80, 407.
28 D. R. Ifa, A. U. Jackson, G. Paglia and R. G. Cooks, Anal. Bioanal.
Chem., 2009.
29 O. K. Yoon, M. D. Robbins, I. A. Zuleta, G. K. Barbula and
R. N. Zare, Anal. Chem., 2008, 80, 8299.
30 J. J. Corr, P. Kovarik, B. B. Schneider, J. Hendrikse, A. Loboda and
T. R. Covey, J. Am. Soc. Mass Spectrom., 2006, 17, 1129.
31 J. C. Jurchen, S. S. Rubakhin and J. V. Sweedler, J. Am. Soc. Mass
Spectrom., 2005, 16, 1654.
32 O. Jardin-Mathe, D. Bonnel, J. Franck, M. Wisztorski, E. Macagno,
I. Fournier and M. Salzet, J. Proteomics, 2008, 71, 332.
33 H.M. B. Spengler, R. Kaufmann, in Proceedings of the 42nd ASMS
Conference on Mass Spectrometry and Allied Topics, Chicago,
Illinois, 1994, p. 1041.
34 S. S. Rubakhin, J. C. Jurchen, E. B. Monroe and J. V. Sweedler, Drug
Discovery Today, 2005, 10, 823.
35 M. L. Reyzer and R. M. Caprioli, J. Proteome Res., 2005, 4, 1138.
This journal is The Royal Society of Chemistry 2009 Nat. Prod. Rep., 2009, 26, 15211534 | 1533
36 M. Wisztorski, D. Croix, E. Macagno, I. Fournier and M. Salzet,
Developmental Neurobiology, 2008, 68, 845.
37 E. Esquenazi, C. Coates, L. Simmons, D. Gonzalez, W. H. Gerwick
and P. C. Dorrestein, Mol. BioSyst., 2008, 4, 562.
38 T. L. Simmons, R. C. Coates, B. R. Clark, N. Engene, D. Gonzalez,
E. Esquenazi, P. C. Dorrestein and W. H. Gerwick, Proc. Natl. Acad.
Sci. U. S. A., 2008, 105, 4587.
39 T. Genji, S. Fukuzawa and K. Tachibana, Mar. Biotechnol., 2009,
DOI: 10.1007/s10126-009-9202-5.
40 Y. L. Yang, Y. Xu, P. Straight and P. C. Dorrestein, accepted,
embargo date 8th November 2009.
41 D. M. G. Anderson, V. A. Carolan, S. Crosland, K. R. Sharples and
M. R. Clench, Rapid Commun. Mass Spectrom., 2009, 23, 1321.
42 R. Shroff, F. Vergara, A. Muck, A. Svatos and J. Gershenzon, Proc.
Natl. Acad. Sci. U. S. A., 2008, 105, 6196.
43 S. W. Cha, H. Zhang, H. I. Ilarslan, E. S. Wurtele, L. Brachova,
B. J. Nikolau and E. S. Yeung, Plant J., 2008, 55, 348.
44 W. Wu, Z. T. Liang, Z. Z. Zha and Z. W. Cai, J. Mass Spectrom.,
2007, 42, 58.
45 S. Robinson, K. Warburton, M. Seymour, M. Clench and J. Thomas-
Oates, New Phytol., 2007, 173, 438.
46 J. A. Hankin, R. M. Barkley and R. C. Murphy, J. Am. Soc. Mass
Spectrom., 2007, 18, 1646.
47 T. W. Jaskolla, M. Karas, U. Roth, K. Steinert, C. Menzel and
K. Reihs, J. Am. Soc. Mass Spectrom., 2009, 20, 1104.
48 S. M. Puolitaival, K. E. Burnum, D. S. Cornett and R. M. Caprioli,
J. Am. Soc. Mass Spectrom., 2008, 19, 882.
49 L. Molin, S. Cristoni, S. Crotti, L. R. Bernardi, R. Seraglia and
P. Traldi, J. Mass Spectrom., 2008, 43, 1512.
50 R. Shroff, L. Rulisek, J. Doubsky and A. Svatos, Proc. Natl. Acad.
Sci. U. S. A., 2009, 106, 10092.
51 Q. Liu, Y. S. Xiao, C. Pagan-Miranda, Y. M. Chiu and L. He, J. Am.
Soc. Mass Spectrom., 2009, 20, 80.
52 S. D. Hanton, T. M. McEvoy and J. R. Stets, J. Am. Soc. Mass
Spectrom., 2008, 19, 874.
53 Y. Sugiura, S. Shimma and M. Setou, Anal. Chem., 2006, 78, 8227.
54 Q. Liu, Z. Guo and L. He, Anal. Chem., 2007, 79, 3535.
55 D. S. Bhakuni and D. S. Rawat, Bioactive marine natural products,
Springer; Anamaya, Dordrecht, New Delhi, 2005.
56 J. B. McClintock and B. J. Baker, Marine chemical ecology, CRC
Press, Boca Raton, Fla., 2001.
57 G. M. Konig, S. Kehraus, S. F. Seibert, A. Abdel-Lateff and
D. Muller, ChemBioChem, 2006, 7, 229.
58 M. G. Haygood, E. W. Schmidt, S. K. Davidson and D. J. Faulkner,
J. Mol. Microbiol. Biotechnol., 1999, 1, 33.
59 J. Piel, Nat. Prod. Rep., 2004, 21, 519.
60 J. Piel, Nat. Prod. Rep., 2009, 26, 338.
61 C. E. Salomon, T. Deerinck, M. H. Ellisman and D. J. Faulkner, Mar.
Biol., 2001, 139, 313.
62 E. N. Tokareva, P. Fardim, A. Pranovich, H. P. Fagerholm,
G. Daniel and B. Holmbom, Appl. Surf. Sci., 2007, 253, 7569.
63 R. Metzner, H. U. Schneider, U. Breuer and W. H. Schroeder, Plant
Physiol., 2008, 147, 1774.
64 S. Moco, B. Schneider and J. Vervoort, J. Proteome Res., 2009, 8,
1694.
65 L. E. Quadri, P. H. Weinreb, M. Lei, M. M. Nakano, P. Zuber and
C. T. Walsh, Biochemistry (Mosc)., 1998, 37, 1585.
66 G. Steinborn, M. R. Hajirezaei and J. Hofemeister, Arch. Microbiol.,
2005, 183, 71.
67 B. Spengler and M. Hubert, J. Am. Soc. Mass Spectrom., 2002, 13,
735.
68 D. S. Peterson, Mass Spectrom. Rev., 2007, 26, 19.
69 R. Knochenmuss and R. Zenobi, Chem. Rev., 2003, 103, 441.
70 M. C. Wood, D. K. Busby and P. B. Farnsworth, Anal. Chem., 2009,
81, 6407.
71 A. B. Costa and R. G. Cooks, Chem. Phys. Lett., 2008, 464, 1.
72 Z. Takats, J. M. Wiseman, B. Gologan and R. G. Cooks, Science,
2004, 306, 471.
73 Z. Takats, J. M. Wiseman and R. G. Cooks, J. Mass Spectrom., 2005,
40, 1261.
74 A. L. Lane, L. Nyadong, A. S. Galhena, T. L. Shearer, E. P. Stout,
R. M. Parry, M. Kwasnik, M. D. Wang, M. E. Hay,
F. M. Fernandez and J. Kubanek, Proc. Natl. Acad. Sci. U. S. A.,
2009, 106, 7314.
75 L. Nyadong, E. G. Hohenstein, A. Galhena, A. L. Lane, J. Kubanek,
C. D. Sherrill and F. M. Fernandez, Anal. Bioanal. Chem., 2009, 394,
245.
76 H. Liebl, J. Appl. Phys., 1967, 38, 5277.
77 D. Debois, K. Hamze, V. Guerineau, J. P. Le Caer, I. B. Holland,
P. Lopes, J. Ouazzani, S. J. Seror, A. Brunelle and O. Laprevote,
Proteomics, 2008, 8, 3682.
78 S. Vaidyanathan, J. S. Fletcher, N. P. Lockyer and J. C. Vickerman,
Appl. Surf. Sci., 2008, 255, 922.
79 S. Vaidyanathan, J. S. Fietcher, R. Goodacre, N. P. Lockyer,
J. Mickleeld and J. C. Vickerman, Anal. Chem., 2008, 80, 1942.
80 J. A. Finzi-Hart, J. Pett-Ridge, P. K. Weber, R. Popa, S. J. Fallon,
T. Gunderson, I. D. Hutcheon, K. H. Nealson and D. G. Capone,
Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 6345.
81 K. Saito, T. Mitsutani, T. Imai, Y. Matsushita and K. Fukushima,
Anal. Chem., 2008, 80, 1552.
82 D. J. Newman and G. M. Cragg, J. Nat. Prod., 2007, 70, 461.
83 G. M. Cragg, P. G. Grothaus and D. J. Newman, Chem. Rev., 2009,
109, 3012.
84 J. A. McLean, W. B. Ridenour and R. M. Caprioli, J. Mass
Spectrom., 2007, 42, 1099.
85 A. B. Kanu, P. Dwivedi, M. Tam, L. Matz and H. H. Hill, J. Mass
Spectrom., 2008, 43, 1.
86 A. Carado, M. K. Passarelli, J. Kozole, J. E. Wingate, N. Winograd
and A. V. Loboda, Anal. Chem., 2008, 80, 7921.
87 D. S. Cornett, S. L. Frappier and R. M. Caprioli, Anal. Chem., 2008,
80, 5648.
88 Q. Z. Hu, N. Talaty, R. J. Noll and R. G. Cooks, Rapid Commun.
Mass Spectrom., 2006, 20, 3403.
89 K. Strupat, V. Kovtoun, H. Bui, R. Viner, G. Stafford and
S. Horning, J. Am. Soc. Mass Spectrom., 2009, 20, 1451.
90 W. T. Liu, J. Ng, D. Meluzzi, N. Bandeira, M. Gutierrez, T. L. Simmons,
A. W. Schultz, R. G. Linington, B. S. Moore, W. H. Gerwick,
P. A. Pevzner and P. C. Dorrestein, Anal. Chem., 2009, 81, 4200.
91 J. Ng, N. Bandeira, W. T. Liu, M. Ghassemian, T. L. Simmons,
W. Gerwick, R. Linington, P. C. Dorrestein and P. Pevzner, Nat.
Methods, 2009, 6, 596.
92 X. Feng, A. S. Ratnayake, R. D. Charan, J. E. Janso, V. S. Bernan,
G. Schlingmann, H. He, M. Tischler, F. E. Koehn and
G. T. Carter, Bioorg. Med. Chem., 2009, 17, 2154.
1534 | Nat. Prod. Rep., 2009, 26, 15211534 This journal is The Royal Society of Chemistry 2009