2010 npr imaging mass spec of natural products.pdf

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Imaging mass spectrometry of natural products

Eduardo Esquenazi,ac Yu-Liang Yang,a Jeramie Watrous,ab William H. Gerwickac

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 profiles 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)

versusion 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 predefined 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 refinement 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, fluorescent analog, or antibody followed by

visual analysis by autoradiography, fluorescence imaging or

immunofluorescence, respectively.1722 Mass spectrometry,

including IMS, can be performed on any substrate provided that

the molecules ionize well enough and are present in sufficient

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 fluorescence, 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 thisHighlightarticle we want to emphasize the application ofIMS to the investigation of natural products, defined here as

metabolites that are directly observed from living organisms that

possess biological functions (e.g. polyketides, nonribosomal

peptides, alkaloids, lantibiotics, microcins, flavinoids, 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

workflow. The first 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

aSkaggs School of Pharmacy and Pharmaceutical Sciences, University ofCalifornia, San Diego, USA. E-mail: [email protected] of Pharmacology, Chemistry and Biochemistry, Universityof California, San Diego, USAcCenter for Marine Biotechnology and Biomedicine, Scripps Institution ofOceanography, 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


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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 workflow is data acquisition. In this process a user-

controlled stage containing the prepared sample is moved in

a defined 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 themethod 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 beamEduardo 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 jointmember 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 Officer 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 analyticalresearch 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 thesupervision 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


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Fig. 1 Overview of imaging mass spectrometry. (A) A schematic outline of the workflow 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 specific 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 traditionalm/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/z511, [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 filaments (B, C). Image C is adapted from

reference 38 with permission.



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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 colorimage 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 defined 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 eachcontain 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 benefit

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, arealso now equipped with xy control. Second, a visualization

software package that allows specific 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 final

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 workflow: 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 Scientific. 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 assetto 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 file 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 arecapable 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-flight (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 configuration 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 cyclotronresonance (ICR) and other types of detector. With so many

possibilities, the field 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



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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 cryosectionedDysidea herbaceasponge with the region that was subjected to npMALDI-I highlighted. (b) Epifluorescence of the sponge

at 590 nm. (c) Epifluorescence ofD. herbaceathin section at 420 nm. (d) MALDI image ofm/z530. This mass and the complex molecular ion isotope cluster

suggest its identity as the hexachlorinated peptide 13-demethylisodysidenin. (e) Autofluorescence (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/z1028. (g) Autofluorescence (420 nm) image overlaid with

m/z530 (red) andm/z1028 (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/z492 0.25), respectively, obtained from mass data measured on the next to last leaf in (a). (d) A scatter plot of 4MSOBvs.I3M. The correspondence with

a line of slopey xshows the extent to which the two compounds co-occur. Scales are in false color intensity (0255) for bothxandyaxes. (e) A scatter plot

of 4MSOBvs. 8MSOO. Images B and C are adapted from references 38 and 42, Copyright (2009) National Academy of Sciences, U.S.A., with permission.



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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 workflow 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 sufficiently

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 newlyformed 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 efficiently,

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 andallowing it to dry, sublimation, applying with a fine 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 specific 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 lowerm/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 (106 torr) inside the

mass spectrometer. At this point, the area of the raster (spot

or continuous) is defined 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 isablated, 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 reflect 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 theanalysis of marine natural products, filamentous and non-fila-

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

profiling to determine the spatial distribution of a marine

natural product, norzoanthamine in the zoanthid Zoanthussp.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 atleast 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-inflammatory 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 utilizingMALDI imaging (using the spot raster method) the spatial

distribution of metabolites in a cross section ofDysidea herbacea,

a marine sponge collected in Papua New Guinea, revealed

a staggering number of different metabolites, some with the

fingerprint 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 epifluorescence, visualization of the



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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 identification 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 toimportant 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

exemplifies 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 feedingdeterrents, 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 sufficient 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 efficacy 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 specific 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 matrixand 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 fine 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 ofBacillus subtilis 3610 and

Streptomyces coelicolorA3(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. subtilisand prodiginine (m/z 392), calcium dependent antibi-otic (CDA,m/z1536) and the morphogen SapB (m/z2027) 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 verified in an independent experiment

where purified surfactin was applied to the agar surface next to

S. coelicolorand 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 theirmorphology but they also silence the defense mechanisms of

neighboring organisms. To confirm 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

significantly increased (Fig. 3B-1).40 Using this agar-based IMS

approach, time-dependent correlations can also be made. For

example, in an effort to find a candidate prodiginine inducer

secreted by B. subtilis, an sfp deletion mutant ofB. subtilis wasapplied 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 them/z 655 ion contacts the

colony of S. coelicolor, it begins to generate the red pigment

prodiginine. Our hypothesis for this scenario is that them/z655

ion is secreted by B. subtilisand 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 purified 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 ofIMS 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.



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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 film 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 ionsas 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 betweenB. subtilis(WTvs.bac-/sfp-) andS. coelicolor. (2) A distance time course of the candidate elicitor of prodiginine,m/z655,

observed surrounding theB. subtilis colony.



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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

significant challenges in its application to natural product

research. Perhaps the most significant issue impacting the use of

MALDI in natural product research is that the matrix itself is

composed of small molecules and associated clusters that aresimilar in size to many natural products of interest (typically

below 1000 Da). In some cases, this can lead to considerable

difficulty in determining whether a specific 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 confirm 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 profile. 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 ofa signal to a specific area of the sample increases confidence 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 sufficient 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 issuccessful 8090% of the time, but has yet to be successful

applied to the imaging of cyanobacterial filaments.

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 ofthe area being sampled. For example, a space of 1 mm2 has about

106 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

field 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. Briefly, a charged solvent spray,

aimed directly at a specific 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 massspectrometer (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 ofC. serratussurface, 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] onC. 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.



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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 predefined xy

direction under the DESI ionization source. To date, DESIimaging 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 recentlyappeared which confirm 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 pathogenLindra 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 exemplifies 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 instrumentalparameters, 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 flow

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 inefficient 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, reflecting the

current technical challenges in the detection of these ions.

However, the development of DESIand its application to imaging

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 difficult to observe by

MALDI, and provides a method which allows the analysis ofsurface 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 configuration 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 first demonstrated in 1910 byJ. J. Thompson and subsequently independently developed as an

instrument in the 1960s by two groups, Liebl and Herzog76 and

Raimond Castaing.1 It has been widely used to interrogate the

composition of surfaces and thin films and thus has been a key

player in material science and geophysics. SIMS is used in

various other fields 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-flight

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, especiallyfor 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 C60+ 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-defined 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 focusedion 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 C60+ 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



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the center and edges of the bacterial colonies (Fig. 5B). Although

the SIMS image of the colony, accomplished with a Bi3+ 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 bySIMS.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 findings

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 significant 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 spacebetween cells, suggesting the secretion of peptides or proteins into

themedium by theculture. 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 first

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 requiresa flat surface. Biological samples are not usually flat, 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 flat 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 byB. subtiliswere 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 C60+ 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 cells79 or an ammonia gradient

observed between heterocysts of filamentous 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 bacteriumB. subtilis (168 sfp+) transferred onto a silicon

wafer. The color scale on the right indicates the relative amounts ofsurfactin 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.



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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 tolaboratories that build or develop mass spectrometry tools.

Moreover, the significant 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 rangecompared 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 IMSholds. 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

significant advances in IMS approaches, in part mediated byimproved 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 AnalyzersaSensitivity/resolution Limitation

Natural products observedwith the different IMSapproachesb

MALDI Sample prep. is critical andan art. The choices ofmatrix and the methodsfor covering samples withmatrix are trial and error.

TOF, ion trap, LTQ,Q-TOF, FT-ICR,Orbitrap

fmolzmol/10100 mm

Matrix signals mayinterfere with the lowm/z region; sample isunder high vacuum.

Nonribosomal Peptides: 13-demethylisodysidenin,38

CDA, surfactins,plipastatins,40

yanucamide.37

Hybrid Peptides/

Polyketides: curacinA,37,38 jamaicamide Aand B,37,38 prodiginine,40

viridamides.37

Flavonoids: kaempferol,43

quercetin,43 quercetin-3-O-rhamnoside43

Alkaloids:Norzoanthamine39

Lantibiotics: themorphogen SapB,40

subtilosin40

DESI Sample prep. is minimaland works under ambientconditions. Instrumentparameters such as spray

angle, spray pressure andrelative geometries needto be optimized for eachsample.

TOF, ion trap, LTQ,QTOF, FT-ICR,Orbitrap

fmolpmol/40400 mm

At the present time limitedsurfaces can be analyzed.For example DESIimaging on solid bacterial

cultures has not beenachieved. DESI has ananalyte washing effect.

Polyketides:bromophycolide A/B,74

bromophycolide E74

SIMS Minimal prep. but samplesurface needs to beespecially clean and flat.

TOF, TRIFT Varies withm/z/100 nm100 mm

Low sensitivity for highm/z (>1000) ions dueto fragmentation; C60

+,Au+ secondary ions hasimproved this limitation.Sample is under highvacuum.

Nonribosomal Peptides:surfactin77

Hybrid Peptide/polyketide:prodiginine78,79

Polyketide: actinorhodin79

Terpenes: hinokione,81

hinokiol81

Lignanoids: hinokinin,81

hinokiresinol81

a Analyzers in bold have been applied to imaging of natural products. b This list is limited to natural products detected by imaging.



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1534 | Nat Prod Rep 2009 26 15211534 This journal is The Royal Society of Chemistry 2009

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