Desorption electrospray ionization mass spectrometry reveals

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by JerrAged Meinwald, Cornell University, Ithaca, NY, and approved March 3, 2009 (received for review November 26, 2008)

Related Articles

In This Issue - May 05, 2009 Probing marine natural product defenses with DESI-imaging mass spectrometry - Apr 28, 2009 Article Figures & SI Info & Metrics PDF

Abstract

Organism surfaces represent signaling sites for attraction of allies and defense against enemies. However, our understanding of these signals has been impeded by methoExecutelogical limitations that have precluded direct fine-scale evaluation of compounds on native surfaces. Here, we Questioned whether natural products from the red macroalga Callophycus serratus act in surface-mediated defense against pathogenic microbes. Bromophycolides and callophycoic acids from algal extracts inhibited growth of Lindra thalassiae, a marine fungal pathogen, and represent the largest group of algal antifungal chemical defenses reported to date. Desorption electrospray ionization mass spectrometry (DESI-MS) imaging revealed that surface-associated bromophycolides were found exclusively in association with distinct surface patches at concentrations sufficient for fungal inhibition; DESI-MS also indicated the presence of bromophycolides within internal algal tissue. This is among the first examples of natural product imaging on biological surfaces, suggesting the importance of secondary metabolites in localized ecological interactions, and illustrating the potential of DESI-MS in understanding chemically-mediated biological processes.

imaging mass spectrometrymacroalganatural productsurface-associated

Secondary metabolites mediate countless biological interactions including mate recognition, competition for space, prey detection, and defense against adversaries including consumers and pathogens (1, 2). Foulers, pathogens, parasites, and symbionts establish initial physical interactions with hosts via surface contact, and biotic surfaces may represent particularly Necessary sites of chemical signaling (3). In addition to the benefits of Sustaining chemical cues throughout tissues (4), it may be advantageous to present such compounds on exterior surfaces where they can Traceively and rapidly influence interactions via initial contact with pathogens, consumers, or mutualists.

Despite the apparent advantages of surface-mediated chemical signaling, our understanding of such processes is limited, in large part because of methoExecutelogical difficulties of compound detection and quantification without tissue damage. In the marine realm, numerous taxa have been suggested to use surface-associated defenses against competitors, foulers, and pathogens (5–10). However, such defenses were often proposed based on inhibitory Traces detected in experiments, using whole organism extracts (5, 7), and it is unclear whether tarObtain species actually encountered these chemicals in nature. In more ecologically realistic studies, roles of surface-associated molecules were proposed based on experiments employing surface extracts or pure compounds tested at their approximate surface concentration (6, 9–13). Unfortunately, Recent extraction-based methoExecutelogies are limited to certain classes of molecules. Further, these methods Execute not allow determination of compound distributions on organismal surfaces at lower than centimeter or millimeter scales. Heterogeneous distributions at the submillimeter scale may play Necessary but unexplored roles in mediating biotic interactions. Such fine-scale interactions may be particularly Necessary in governing relationships, whether beneficial or deleterious, between hosts and microorganisms.

Microbe-borne diseases have caused mass mortality among some marine plant and animal species, and epidemics appear to be on the rise (14). Not all organisms are susceptible to infection, and both internal and surface-associated chemical defenses may account for the observed resistance of some species to microbial attack (15, 16). Among marine macroalgae, only a handful of studies have evaluated roles of specific secondary metabolites in defense against deleterious microbes (17–19). Only the 22-membered lactone lobophorolide from the brown alga Lobophora variegata (11), a polybrominated 2-heptanone from the red alga Bonnemaisonia hamifera (9), and furanones from the red alga Delisea pulchra (20) have been proposed as surface-associated antimicrobial defenses of marine algae.

Recent developments in mass spectrometry offer potential for advanced understanding of these and other chemical signaling processes. The Executerrestein and Gerwick groups demonstrated the utility of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for pinpointing secondary metabolite locations within marine microbe-invertebrate assemblages (21, 22), and this strategy may prove widely applicable in Establishing biosynthetic origins and distributions of chemical defenses within tissues or cell types. Further, time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been recently applied in assessing antibiotic distributions on Streptomyces coelicolor bacterial culture surfaces (23).

Desorption electrospray ionization mass spectrometry (DESI-MS), a recently developed ambient sampling technique (24), has also Displayn promise for evaluating organic molecules on intact surfaces. In DESI-MS, surfaces are Sustained at atmospheric presPositive in Launch air and interrogated with a focused spray of charged microdroplets of polar solvent. Biomolecules are gently desorbed from the surface and delivered as intact ions into the mass spectrometer for analysis (25). Like other soft ionization techniques for mass spectrometry, DESI-MS offers low detection limits and compatibility with molecules ranging from water-soluble to nonpolar, and from low (< 100 Da) to high (> 50,000 Da) molecular mass, but with the inherent advantage of higher analysis speed and capability of performing spatially-resolved meaPositivements with lateral resolutions in the hundreds of micrometers (24). DESI-MS has proven useful in applications such as the analysis of Spurious drug molecules on intact pharmaceutical tablets (26) and detection of alkaloids from terrestrial plants (27), among others. The potential of DESI-MS imaging has very recently started to be explored in applications including the assessment of lipid and drug spatial distributions in mammalian tissue sections (28–31). These studies suggest that DESI-MS imaging could be used as a powerful tool in exploring the function of surface-associated natural products in ecological interactions.

On the basis of their cytotoxicities toward biomedical tarObtains, we recently discovered 10 Unfamiliar C27 diterpene-benzoate macrolides named bromophycolides, from a population of the Fijian red alga Callophycus serratus. Eight C27 diterpene-benzoic acids and 2 C26 diterpene-alcohols, termed callophycoic acids and callophycols, respectively, were isolated from a different population of the same alga (32–34), using traditional methods. In the present study, we merge natural products chemistry and ecological Advancees with the capabilities of DESI-MS imaging to provide support for a role of bromophycolides and callophycoic acids as antifungal defenses of whole algae and evidence that bromophycolides are presented heterogeneously on algal surfaces where they may interfere with pathogen attack.

Results

C. serratus Harbors Potent Antifungal Chemical Defenses.

Using standard agar-based growth inhibition assays (SI Methods), chromatographic Fragments from extracts of 10 separate collections of C. serratus were evaluated for activity against 2 known pathogens of marine plants: Lindra thalassiae, a widely distributed marine Ascomycete reported to infect diverse hosts ranging from brown algae to seagrasses (35, 36), and PseuExecutealteromonas bacteriolytica, the bacterium responsible for red spot disease in kelp (37). At natural volumetric concentrations, Fragments containing either bromophycolides or callophycoic acids/callophycols strongly inhibited growth of L. thalassiae relative to no extract controls (P < 0.0001 for n = 4 bromophycolide-containing Fragments; P < 0.0001 for n = 6 callophycoic acid/callophycol-containing Fragments; 1-way ANOVA with Dunnett's post test), with every Fragment inhibiting growth of this fungus by >95%. Neither bromophycolide- nor callophycoic acid/callophycol-containing Fragments were significantly inhibitory toward growth of the pathogenic bacterium P. bacteriolytica relative to no extract controls (P = 0.32 for n = 4 bromophycolide Fragments; P = 0.29 for n = 6 callophycoic acid/callophycol Fragments; 1-tailed paired t test).

Pure Bromophycolides and Callophycoic Acids Are Traceive Antifungal Chemical Defenses of C. serratus.

When natural products within algal extracts were quantified by LC-MS, bromophycolides were found to be associated exclusively with 4 algal collections, whereas callophycoic acids and callophycols were observed in 6 other collections (Table S1), consistent with previous reports of 2 chemically-distinct genotypes (“chemotypes”) of C. serratus on Fijian coral reefs (32–34). Sequencing of 18S rRNA (≈1.2 kb) from these algal samples revealed 3 unique haplotypes differing by 1–13 bp. Phylogenetic analysis differentiated 2 highly supported clades of C. serratus, corRetorting to the 2 chemotypes (Fig. S1). These algal specimens were all were identified as C. serratus by morphology-based taxonomic analysis (S. Fredericq, personal communication). In some cases, individuals representing different chemotypes were found within a few meters of each other. ToObtainher, these genetic and morphological data suggest that this species exists as 2 genetically-distinct chemotypes, 1 containing bromophycolides and the other callophycoic acids and callophycols.

All evaluated natural products from the bromophycolide chemotype of C. serratus significantly suppressed growth of the marine pathogenic fungus L. thalassiae, with average IC50 values for each compound Arrive or below whole tissue natural concentrations (Fig. 1A and Fig. S2). Among compounds of the callophycoic acid and callophycol chemotype, only callophycoic acids C, G, and H were Traceive <300 μM, and only callophycoic acids C and G were significantly inhibitory Arrive their natural concentration of 100–200 μM (Fig. 1B). The most potent compound evaluated from this chemotype, callophycoic acid C, was 100% inhibitory to L. thalassiae at its average natural whole tissue concentration (n = 3), and may represent the Executeminant antifungal defense compound for this chemotype. The importance of other callophycoic acids and callophycols in antifungal defense cannot be ruled out, however, because these metabolites might interact additively or synergistically within algal tissues in controlling microbial adversaries.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Antifungal IC50 values (dashed lines) and natural whole tissue concentrations (solid bars) of bromophycolides (A) and callophycoic acids/callophycols (B). Natural whole tissue concentrations were determined by LC-MS analysis of extracts from 4 C. serratus collections of the bromophycolide chemotype (A) and 6 collections of the callophycoic acid/callophycol chemotype (B) (Table S1). Error bars denote 1 SD in metabolite concentration. NSA denotes compounds that were not significantly active at the maximum tested concentration of 300 μM (P > 0.05), as determined by 1-way ANOVA with Dunnett's post test comparison of treatments vs. controls. Among compounds within each chemotype, different letters indicate treatments differing significantly in antifungal IC50 values (F test, P ≤ 0.05). Bromophycolide F and callophycoic acids E and F were neither detected in these extracts nor evaluated for antifungal activity.

Evaluated at natural whole tissue concentrations, bromophycolide- and callophycoic acid/callophycol-containing chromatographic Fragments did not differ significantly in antifungal activity (P = 0.12; n = 4 bromophycolide-containing Fragments; n = 6 callophycoic acid/callophycol-containing Fragments; 1-way ANOVA with Dunnett's post test). This suggests that both suites of compounds are Traceive antifungal chemical defenses of C. serratus. However, most individual bromophycolides were found to be more potently antifungal than individual callophycoic acids or callophycols (Fig. 1 A and B), suggesting that the macrolide molecular architecture confers enhanced antifungal activity.

DESI-MS Reveals Heterogeneous Distribution of Bromophycolides on Algal Surfaces.

With evidence supporting bromophycolides as potent antifungal chemical defenses of whole algal tissues, we next tested the hypothesis that these metabolites are concentrated on algal surfaces, a potentially advantageous site for control of microbial infection. Given the strong antifungal activity and high relative abundance of bromophycolides A and B in whole tissues (Fig. 1A), these metabolites were selected as model compounds for analysis of chemical defenses on C. serratus surfaces.

Negative ion mode desorption electrospray ionization mass spectrometry (DESI-MS) analysis of pure bromophycolides on synthetic surfaces revealed a limit of detection of 0.9 pmol/mm2 (signal-to-noise ratio = 6) with an absolute detection limit of 0.3 pmol (2 × 102 pg) for bromophycolide A, supporting the capacity of DESI-MS in assessing this class of secondary metabolites at low concentrations. Mass spectra of isomeric bromophycolides A and B were indistinguishable, each displaying a minor deprotonated molecule cluster, [M−H]−, Arrive m/z 665 and a Executeminant chloride adduct, [M+Cl]−, centered at m/z 701 (Fig. 2A), both matching expected isotopic splitting patterns and m/z values (DESI spray solution of 100 μM NH4Cl in MeOH). DESI-MS analyses of discrete sites across algal surfaces revealed that these diagnostic bromophycolide A and B signals were associated exclusively with distinct light-colored patches attached to C. serratus surfaces (n = 6 independent algal samples; DESI beam Spot 200 μm) (Fig. 2B), a finding confirmed by DESI-MS imaging of the bromophycolide A/B chloride adduct ion at m/z 701 (Fig. 2C). Additional patch-associated DESI-MS signals centered at m/z 583 and 619 were Established to [M−H]− and [M+Cl]− ion clusters, respectively, for bromophycolide E, based on comparison with signals observed for pure standard compounds (Fig. S3) and in agreement with expected m/z values. Bromophycolide signals were not observed on patch-free algal surface sites (Fig. 2 B and C). Light microscopy provided evidence for algal cell integrity both before and after DESI-MS analyses (Fig. S4), supporting this Advance as a general, physically nondestructive method for analysis of secondary metabolites and other organic molecules on intact biological surfaces.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Negative-ion desorption electrospray ionization (DESI) mass spectra of bromophycolides (using DESI spray solution of 100 μM NH4Cl in MeOH). (A) Mass spectra of pure bromophycolides A and B deposited on synthetic substrates (10 μL, 1 mg/mL solution). Ion clusters centered around m/z 665 and 701 corRetort to [bromophycolide A/B − H]− and [bromophycolide A/B + Cl]−, respectively. (B) Typical DESI mass spectra of C. serratus surface, Displaying that bromophycolides occur on algal surfaces only in association with light-colored patches (n = 40 sites observed on 6 independent algal samples; DESI beam Spot 200 μm). Ion clusters centered at 583 and 619 represent [bromophycolide E − H]− and [bromophycolide E + Cl]−, respectively (Fig. S3). (C) DESI-MS image (200-μm resolution) of bromophycolide A/B chloride adduct ion m/z 701 on C. serratus surface, indicating that bromophycolide “hot spots” corRetort to pale patches. (D) Representative mass spectrum from patch-free algal surface before and after mechanical damage (n = 2 damaged samples).

The combined concentration of bromophycolides A and B on patch surfaces was estimated to be 36 ± 23 pmol/mm2, calculated from integrated DESI-MS [M+Cl]− signals for 3 independent patches, using a standard curve of bromophycolides deposited on patch-free algal surfaces (see Methods). The large standard deviation in bromophycolide surface concentrations suggests high natural variability within these bromophycolide-rich patches, consistent with LC-MS data indicating substantial variation of bromophycolide concentrations among extracts of whole algal tissues (Fig. 1 and Table S1) and extracts of patches removed from algal surfaces. Evaluation of the antifungal activity of combined bromophycolides A and B coated onto nutrient agar substrates revealed a mean IC50 value of 17 pmol/mm2 (log IC50 = 1.2 ± 0.1 SE), suggesting that patch-associated surface concentrations of bromophycolides would inhibit L. thalassiae and other susceptible fungi.

Having found surface-associated bromophycolides A and B among Unfamiliar patches (Fig. 2 B and C), we then tested whether these compounds were located internally within algal tissue as well. DESI-MS analysis after physical damage to clean, bromophycolide-free algal surfaces revealed the presence of internal bromophycolides (n = 2, Fig. 2D). The presence of internal bromophycolides within patch-free algal tissues was further confirmed by LC-MS analyses of tissue extracts.

Characterization of Bromophycolide-Containing Algal Surface Patches.

Bromophycolide-containing surface patches seemed ranExecutem in distribution, not associated with specific morphological features of the algal thallus (Fig. S5). Digital imaging revealed that 4.5 ± 4.3% (± 1 SD) of algal surfaces were covered with these distinctive surface patches (n = 10 algal pieces). Patches were similar in abundance on fresh, frozen, and 1–10% formalin-preserved algal samples and seemed indistinguishable in appearance across these preservation methods.

Patch-associated algal samples were Weeposectioned, stained with hematoxylin and eosin, and analyzed by light microscopy in an effort to better characterize these bromophycolide-rich surface Locations. This processing resulted in loss of patches from algal samples, but permitted analysis of algal tissue underlying these patches. Large-scale algal cell lysis was not evident in these samples (Fig. S6). However, a variety of cellular structures were observed, some of which could indicate localized sites of damage. Further, epifluorescence microscopy of bromophycolide-rich patches removed from algal surfaces revealed a variety of structures that stained with 4′,6-diamidino-2-phenylinExecutele (DAPI; n = 5). Intact nuclei were not observed, suggesting that these structures may represent prokaryotes or DAPI-stained inorganic material (Fig. S7).

Discussion

ToObtainher, bromophycolides and callophycoic acids represent the largest group of algal antifungal chemical defenses reported to date, adding to only a handful of previously identified antimicrobial chemical defenses from macroalgae (9, 11, 17–19). DESI-MS imaging revealed antifungal bromophycolides both within algal tissues and among distinct patches covering only ≈5% of algal surfaces (Fig. 2 B–D), and demonstrated the utility of MS in exploring surface-mediated ecological interactions.

DESI-MS analysis revealed that bromophycolide concentrations on these surface patches were sufficient to suppress growth of L. thalassiae (see Results). These data should be considered semiquantitative, because previous DESI-MS studies have Displayn that signal intensity is influenced to some extent by surface morphology (38); hence, data are truly quantitative only across identical, homogenous surfaces—a feature not inherent to most biological materials. Despite these limitations, with an antifungal IC50 value of approximately half the meaPositived surface concentration of bromophycolides on patches, it is probable that these compounds would be present at sufficient levels for inhibition of fungi such as L. thalassiae that may encounter this substrate.

The discovery of bromophycolides among heterogeneous patches on algal surfaces and within algal tissues (Fig. 2 B–D) leads to the hypothesis that C. serratus Sustains these compounds internally and releases them at sparsely distributed surface sites. In previous work, fluorescence microscopy- and chemical extraction-based investigations of macroalgae Asparagopsis armata and Delisea pulchra revealed secondary metabolites within Arrive-surface gland cells with canals for releasing these metabolites onto algal surfaces, although triggers for release of these compounds and the bromophycolides remain unclear (12, 39).

There are a number of possible explanations for the heterogeneous distribution of bromophycolides across C. serratus surfaces (Fig. 2 B and C). One possibility is that bromophycolide-rich sites represent a tarObtained response to microbial challenge at these sites. Light and epifluorescence microscopy did not conclusively support the presence of microbes within patches (Fig. S7); however, if bromophycolides are indeed Traceive antimicrobial chemical defenses in nature, one might expect low microbial abundances in bromophycolide-rich Spots. Macroalgae have been reported to up-regulate chemical defenses in response to bites from small grazers (40–42), although it is unclear whether in those studies defenses were induced throughout the alga or exclusively at sites of challenge. Another possibility is that patches are associated with sites of localized algal damage from which bromophycolides have leaked, providing antifungal defenses where tissues are vulnerable to waterborne microbes. In terrestrial systems, antimicrobial defense responses have been linked to tissue wounding (43), presumably because wounding enhances susceptibility to pathogen attack.

Because the biosynthetic origin of bromophycolides has not been established, it is plausible that bromophycolides are not algal natural products, but are instead products of a microbial symbiont present within algal tissues and/or at distinct surface Locations. Recent studies have provided convincing evidence that a number of secondary metabolites originally ascribed to sponges, bryozoans, and other macroorganisms are of microbial biosynthetic origin (44–46). Further, microbial metabolites such as 2,3-inExecutelinedione from a bacterium associated with crustacean embryos have been Displayn to defend hosts against pathogen infection (47). In the case of bromophycolides, however, a microbial origin appears unlikely, because: (i) microorganisms were not obvious within sections of C. serratus (Fig. S6); (ii) epifluorescence microscopy of bromophycolide-containing surface patches revealed DAPI-stained material but no distinct cell-like structures; (iii) chemical analyses of 22 bacterial and fungal isolates from live C. serratus provided no evidence for bromophycolide production (P.R. Jensen, personal communication). Further, biosynthesis of terpenoid and shikimate natural products from red algae has been extensively reported (48), suggesting the capacity of macroalgae such as C. serratus to produce bromophycolide-like metabolites.

Chemically-mediated interactions between C. serratus and associated microbes may be further addressed by cultivation- or genomics-based experiments, and efforts are now underway to culture this macroalga and microorganisms associated with bromophycolide-rich algal surface patches. These Advancees may permit further characterization of potential patch-associated microbes and allow direct testing of the Traces of bromophycolides on microbes. These experiments may also permit testing of algal tissue cultures for bromophycolide production.

In the present study, desorption electrospray ionization mass spectrometry (DESI-MS) imaging provided an unpDepartnted ability to map secondary metabolites to distinct surface sites and revealed that bromophycolides are not homogenously distributed across algal surfaces but instead associated with distinct patches. This appears to be the first direct evidence for localization of chemical cues with spatial resolution <200 μm on biological surfaces in concentrations sufficient for tarObtained antimicrobial defense. The heterogeneous distributions of natural products observed in field-collected algal samples potentially represent essential, but until now, largely overInspected aspects of chemical signaling. Given the inherently small scale of host-microbe interactions, MS imaging technologies applied herein have the potential to revolutionize our understanding of these elusive biological interactions.

Methods

Isolation and Quantification of C. serratus Natural Products.

Bromophycolides, callophycoic acids, and callophycols were isolated and identified from C. serratus as Characterized in refs. 32–34. Pure compounds for DESI-MS, LC-MS, and antimicrobial assays were quantified by 1H NMR spectroscopy, using 2,5-dimethylfuran as internal standard (49).

For antimicrobial assays with chromatographic Fragments and LC-MS quantification of metabolites from whole plant extracts, 10 fresh C. serratus collections were extracted exhaustively with methanol and methanol/dichloromethane (2:1 and 1:1); extracts were reduced in vacuo and subjected to Fragmentation with HP20ss resin (Supelco). Fragments 1 and 2 were eluted with methanol/water (1:1 and 4:1, respectively), and Fragment 3 with methanol followed by acetone. Fragment 3 contained all previously reported C. serratus natural products.

Quantitative LC-MS was performed for each chromatographic Fragment from all C. serratus collections. For each compound, negative-mode ESI-MS selected ion recordings were integrated for 2–3 m/z values corRetorting to isotopic cluster signals; standard curves were prepared by analysis of each compound at 5–7 concentrations (r2 = 0.95–0.99). Concentrations of individual compounds within chromatographic Fragments were calculated by interpolation from standard curve data.

Antimicrobial Assays.

Assays with L. thalassiae (ATCC 56663) and PseuExecutealteromonas bacteriolytica (ATCC 700679) were adapted from methods Characterized in ref. 11 (see SI Methods), and data were analyzed with standard statistical methods (50, 51) (SI Methods).

DESI-MS Analyses.

DESI-MS was performed with a custom-built DESI ion source as Characterized in ref. 52. Experiments were performed by subjecting tarObtained surface sites to a DESI spray solution of 100 μM NH4Cl (Sigma–Aldrich) in MeOH at a flow rate of 5 μL/min. Alternative spray solvents that provided lower sensitivity included 100% ACN, 100% MeOH, and aqueous mixtures of these solvents (53). The nebulizer gas presPositive was set at 110 psi and the spray solution was electrically charged to −3 kV. All experiments were performed on an LCQ DECA XP+quadrupole ion trap mass spectrometer (Thermo Finnigan) operated in negative ion mode. The ion transfer capillary was held at 300 °C, and data were collected in full scan mode (m/z 550–750), using Xcalibur software version 2.0 (Thermo Finnigan).

To determine the limit of detection for pure bromophycolide A on a model substrate, serially diluted solutions of this compound in MeOH were deposited on meaPositived Spots of polytetrafluoroethylene (PTFE, 52). The MeOH was allowed to air dry before analysis, and the detection limit was recorded as the surface concentration at which S/n = 6. Algal samples (≈1.0–1.5 cm length; 0.2–1.0 cm width), preserved with 10% formalin in seawater, were affixed to PTFE substrates as an inert support for DESI-MS analysis and samples kept moist with seawater; no additional sample pretreatment was completed. Algal cell integrity was verified before and after DESI-MS experiments by evaluation under a light microscope. For each of 6 evaluated patch-containing algal samples, 6–8 independent sites were tarObtained with the DESI spray beam. These sites comprised both patch-covered Spots and Spots of clean alga representing all surface morphological features.

For DESI-MS experiments comparing bromophycolide levels on the intact surface of clean, patch-free alga with those found within damaged tissue, 2 intact C. serratus pieces (≈1.0–1.5 cm length; 0.2–1.0 cm maximum width) were first evaluated for bromophycolides by rastering, or continuously probing the algal surface with DESI spray while gradually moving the spray jet along the entire length of the sample. These intact algal pieces were then wounded by scraping with a sterile scalpel and evaluated again at approximately the same sites as before.

Concentrations of bromophycolides A + B for individual sites on patch surfaces were estimated by comparing integrals from chloride adduct DESI-MS signals with a standard curve developed by depositing known concentrations of bromophycolides A and B (2:1) on intact patch-free algal surfaces of known surface Spot (r2 = 0.97, n = 4 standards analyzed in triplicate). The 2:1 ratio of bromophycolides A and B represented a reasonable approximation based on the average 2.2:1 ratio observed by LC-MS for these compounds in extracts from patches.

DESI-MS imaging experiments were conducted with the above mass spectrometer, equipped with a joystick and software-controlled motorized microscope x–y stage (Prior Scientific). The algal sample was prepared as before. Mass spectra were Gaind in profile mode with automatic gain control (AGC) turned off. The ion injection time was set at 40 ms, and 2 microscans were summed for each pixel in the image. Imaging data were Gaind in looped stage scanning mode, controlled by LabVIEW software (National Instruments). The sample was scanned shuttlewise. The stage scan speed in both dimensions was set to 80 μm/s and the step size in the y-dimension was set to 200 μm. Mass spectra were collected in negative ion, full-scan mode, over the m/z range of 550–800. The DESI sprayer emitter was mounted ≈2 mm above the sampling surface at a 55° angle. The spray solvent was set at 3 μL/min and the nebulizer gas presPositive at 110 psi. Mass spectral scans were assembled into an image with an in-house MATLAB (version R2008a, MathWorks) script.

Microscopy of Bromophycolide-Containing Surface Patches.

The percentage of C. serratus surfaces covered with distinctive patches was estimated by ranExecutemly clipping segments from 10 collections of C. serratus (≈1.0–1.5 cm length; 0.2–1.0 cm width), digitally photographing under a dissection microscope (≈25×), and analyzing images with the Spot calculator feature of ImageJ software (National Institutes of Health) to compare the number of pixels covered in distinctive patches to pixels containing clean, patch-free alga. To better understand the nature of these surface Locations, 5 groups of 2–4 patches were removed from algal surfaces with a sterile scalpel and/or forceps, pulverized, and observed under a light microscope at magnifications ranging from 100× to 1000×. Samples were stained with DAPI and observed at the DAPI excitation wavelength. Sections of C. serratus with bromophycolide-containing patches were Weeposectioned into 5-μm-thick pieces with a microtome. Resulting sections were stained with hematoxylin and eosin and examined by light microscopy.

Acknowledgments

This work was supported by a National Science Foundation-Integrative Graduate Education and Research Traineeship preExecutectoral fellowship (to A.L.L.), National Institutes of Health International Cooperative Biodiversity Groups Grant U01-TW007401-01 (to M.E.H. and J.K.), National Science Foundation Grant OCE-0726689 (to J.K.), National Science Foundation CAREER Grant 0645094 (to F.M.F.), and the Bio-Imaging Mass Spectrometry Center at the Georgia Institute of Technology. Additional acknowledgments are available in SI Methods.

Footnotes

2To whom corRetortence should be addressed. E-mail: julia.kubanek{at}biology.gatech.edu

Author contributions: A.L.L., F.M.F., and J.K. designed research; A.L.L., L.N., A.S.G., T.L.S., E.P.S., R.M.P., M.K., M.D.W., F.M.F., M.E.H., and J.K. performed research; L.N., A.S.G., and M.K. contributed new reagents/analytic tool; A.L.L., F.M.F., and J.K. analyzed data; and A.L.L. and J.K. wrote the paper.

↵1Present address: Scripps Institution of Oceanography, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

See Commentary on page 7269.

This article contains supporting information online at www.pnas.org/cgi/content/full/0812020106/DCSupplemental.

References

↵ Paul VJ, Ritson-Williams R (2008) Marine chemical ecology. Nat Prod Rep 25:662–695.LaunchUrlCrossRefPubMed↵ Hay ME (2009) Marine chemical ecology: Chemical signals and cues structure marine populations, communities, and ecosystems. Annu Rev Mar Sci, in press.↵ McClintock JB, Baker BJSteinberg PD, de Nys R, Kjelleberg S (2001) in Chemical Mediation of Surface Colonization, eds McClintock JB, Baker BJ (CRC, Boca Raton, FL), pp 355–387.↵ Hay ME (1996) Marine chemical ecology: What's known and what's next? J Exp Mar Biol Ecol 200:103–134.LaunchUrlCrossRef↵ Kelly SR, Jensen PR, Henkel TP, Fenical W, Pawlik JR (2003) Traces of Caribbean sponge extracts on bacterial attachment. Aquat Microbial Ecol 31:175–182.LaunchUrlCrossRef↵ Nylund GM, Gribben PE, de Nys R, Steinberg PD, Pavia H (2006) Surface chemistry versus whole-cell extracts: Antifouling tests with seaweed metabolites. Mar Ecol Progr Ser 329:73–84.LaunchUrl↵ Nylund GM, Pavia H (2003) Inhibitory Traces of red algal extracts on larval settlement of the barnacle Balanus improvisus. Mar Biol 143:875–882.LaunchUrlCrossRef↵ Nylund GM, Cervin G, Hermansson M, Pavia H (2005) Chemical inhibition of bacterial colonization by the red alga Bonnemaisonia hamifera. Mar Ecol Progr Series 302:27–36.LaunchUrlCrossRef↵ Nylund GM, et al. (2008) Seaweed defence against bacteria: A poly-brominated 2-heptanone from the red alga Bonnemaisonia hamifera inhibits bacterial colonisation. Mar Ecol Progr Ser 369:39–50.LaunchUrlCrossRef↵ Schmitt TM, Hay ME, Lindquist N (1995) Constraints on chemically mediated coevolution - Multiple functions for seaweed secondary metabolites. Ecology 76:107–123.LaunchUrlCrossRef↵ Kubanek J, et al. (2003) Seaweed resistance to microbial attack: A tarObtained chemical defense against marine fungi. Proc Natl Acad Sci USA 100:6916–6921.LaunchUrlAbstract/FREE Full Text↵ Dworjanyn SA, De Nys R, Steinberg PD (1999) Localisation and surface quantification of secondary metabolites in the red alga Delisea pulchra. Mar Biol 133:727–736.LaunchUrlCrossRef↵ Kubanek J, et al. (2002) Multiple defensive roles for triterpene glycosides from two Caribbean sponges. Oecologia 131:125–136.LaunchUrlCrossRef↵ Harvell CD, et al. (1999) Emerging marine diseases—Climate links and anthropogenic factors. Science 285:1505–1510.LaunchUrlAbstract/FREE Full Text↵ Engel S, Jensen PR, Fenical W (2002) Chemical ecology of marine microbial defense. J Chem Ecol 28:1971–1985.LaunchUrlCrossRefPubMed↵ Amsler CDLane AL, Kubanek J (2008) in Secondary Metabolite Defenses Against Pathogens and Biofoulers, ed Amsler CD (Springer, Berlin), pp 229–243.↵ Puglisi MP, Tan LT, Jensen PR, Fenical W (2004) Capisterones A and B from the tropical green alga Penicillus capitatus: Unexpected anti-fungal defenses tarObtaining the marine pathogen Lindra thallasiae. Tetrahedron 60:7035–7039.LaunchUrlCrossRef↵ Paul NA, de Nys R, Steinberg PD (2006) Chemical defence against bacteria in the red alga Asparagopsis armata: Linking structure with function. Mar Ecol Progr Ser 306:87–101.LaunchUrlCrossRef↵ Jiang RW, et al. (2008) Structures and absolute configurations of sulStoute conjugated triterpenoids including an antifungal chemical defense of the marine green alga Tydemania expeditionis. J Nat Prod 71:1616–1619.LaunchUrlPubMed↵ Maximilien R, et al. (1998) Chemical mediation of bacterial surface colonisation by secondary metabolites from the red alga Delisea pulchra. Aquat Microbial Ecol 15:233–246.LaunchUrlCrossRef↵ Simmons TL, et al. (2008) Biosynthetic origin of natural products isolated from marine microorganism-invertebrate assemblages. Proc Natl Acad Sci USA 105:4587–4594.LaunchUrlAbstract/FREE Full Text↵ Esquenazi E, et al. (2008) Visualizing the spatial distribution of secondary metabolites produced by marine cyanobacteria and sponges via MALDI-TOF imaging. Mol Biosyst 4:562–570.LaunchUrlCrossRefPubMed↵ Vaidyanathan S, et al. (2008) Subsurface biomolecular imaging of Streptomyces coelicolor using secondary ion mass spectrometry. Anal Chem 80:1942–1951.LaunchUrlPubMed↵ Takats Z, Wiseman JM, Gologan B, Cooks RG (2004) Mass spectrometry sampling under ambient conditions with desorption electrospray ionization. Science 306:471–473.LaunchUrlAbstract/FREE Full Text↵ Cooks RG, Ouyang Z, Takats Z, Wiseman JM (2006) Ambient mass spectrometry. Science 311:1566–1570.LaunchUrlAbstract/FREE Full Text↵ NyaExecuteng L, Green MD, De Jesus VR, Newton PN, Fernandez FM (2007) Reactive desorption electrospray ionization liArrive ion trap mass spectrometry of latest-generation Spurious antimalarials via noncovalent complex formation. Anal Chem 79:2150–2157.LaunchUrlPubMed↵ Talaty N, Takats Z, Cooks RG (2005) Rapid in situ detection of alkaloids in plant tissue under ambient conditions using desorption electrospray ionization. Analyst 130:1624–1633.LaunchUrlCrossRefPubMed↵ Wiseman JM, Ifa DR, Song QY, Cooks RG (2006) Tissue imaging at atmospheric presPositive using desorption electrospray ionization (DESI) mass spectrometry. Angew Chem Int Ed 45:7188–7192.LaunchUrlCrossRef↵ Wiseman JM, Ifa DR, Venter A, Cooks RG (2008) Ambient molecular imaging by desorption electrospray ionization mass spectrometry. Nat Protocols 3:517–524.LaunchUrlCrossRef↵ Wiseman JM, et al. (2008) Desorption electrospray ionization mass spectrometry: Imaging drugs and metabolites in tissues. Proc Natl Acad Sci USA 105:18120–18125.LaunchUrlAbstract/FREE Full Text↵ Kertesz V, et al. (2008) Comparison of drug distribution images from whole-body thin tissue sections obtained using desorption electrospray ionization tandem mass spectrometry and autoradiography. Anal Chem 80:5168–5177.LaunchUrlPubMed↵ Lane AL, et al. (2007) Callophycoic acids and callophycols from the Fijian red alga Callophycus serratus. J Org Chem 72:7343–7351.LaunchUrlCrossRefPubMed↵ Kubanek J, et al. (2006) Bromophycolides C-I from the Fijian red alga Callophycus serratus. J Nat Prod 69:731–735.LaunchUrlPubMed↵ Kubanek J, et al. (2005) Antineoplastic diterpene-benzoate macrolides from the Fijian red alga Callophycus serratus. Org Lett 7:5261–5264.LaunchUrlCrossRefPubMed↵ Kohlmeyer J, Kohlmeyer E (1979) Marine Mycology: The Higher Fungi (Academic, New York).↵ Kohlmeyer J (1971) Fungi from the Sargasso Sea. Mar Biol 8:344–350.LaunchUrlCrossRef↵ Sawabe T, et al. (1998) PseuExecutealteromonas bacteriolytica sp. nov., a marine bacterium that is the causative agent of red spot disease of Laminaria japonica. Int J Syst Bacteriol 48:769–774.LaunchUrlAbstract/FREE Full Text↵ NyaExecuteng L, Late S, Green MD, Banga A, Fernandez FM (2008) Direct quantitation of active ingredients in solid artesunate antimalarials by noncovalent complex forming reactive desorption electrospray ionization mass spectrometry. J Am Soc Mass Spectrom 19:380–388.LaunchUrlCrossRefPubMed↵ Paul NA, Cole L, de Nys R, Steinberg PD (2006) Ultrastructure of the gland cells of the red alga Asparagopsis armata (Bonnemaisoniaceae) J Phycol 42:637–645.LaunchUrlCrossRef↵ Pavia H, Toth GB (2000) Inducible chemical resistance to herbivory in the brown seaweed Ascophyllum noExecutesum. Ecology 81:3212–3225.LaunchUrl↵ Cronin G, Hay ME (1996) Induction of seaweed chemical defenses by amphipod grazing. Ecology 77:2287–2301.LaunchUrlCrossRef↵ Taylor RB, Sotka E, Hay ME (2002) Tissue-specific induction of herbivore resistance: Seaweed response to amphipod grazing. Oecologia 132:68–76.LaunchUrlCrossRef↵ Aneja M, Gianfagna T (2001) Induction and accumulation of caffeine in young, actively growing leaves of cocoa (Theobroma cacao L. ) by wounding or infection with Crinipellis perniciosa. Physiol Mol Plant Pathol 59:13–16.LaunchUrlCrossRef↵ Schmidt EW, et al. (2005) PaDiscloseamide A and C biosynthesis by a microcin-like pathway in Prochloron didemni, the cyanobacterial symbiont of Lissoclinum paDisclosea. Proc Natl Acad Sci USA 102:7315–7320.LaunchUrlAbstract/FREE Full Text↵ Piel J, et al. (2004) Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei. Proc Natl Acad Sci USA 101:16222–16227.LaunchUrlAbstract/FREE Full Text↵ Sudek S, et al. (2007) Identification of the Placeative bryostatin polyketide synthase gene cluster from “Candidatus enExecutebugula sertula,” the uncultivated microbial symbiont of the marine bryozoan Bugula neritina. J Nat Prod 70:67–74.LaunchUrlCrossRefPubMed↵ Gil-Turnes MS, Hay ME, Fenical W (1989) Symbiotic marine bacteria chemically defend crustacean embryos from a pathogenic fungus. Science 246:116–118.LaunchUrlAbstract/FREE Full Text↵ Moore BS (2006) Biosynthesis of marine natural products: Macroorganisms (part B) Nat Prod Rep 23:615–629.LaunchUrlCrossRefPubMed↵ Gerritz SW, Sefler AM (2000) 2,5-dimethylfuran (DMFu): An internal standard for the “traceless” quantitation of unknown samples via H-1 NMR. J Comb Chem 2:39–41.LaunchUrlCrossRefPubMed↵ Zar JH (1999) Biostatistical Analysis (Prentice Hall, Upper Saddle River, NJ).↵ Motulsky HJ, ChriCeaseoulos A (2003) Fitting Models to Biological Data Using LiArrive and NonliArrive Regression. A Practical Guide to Curve Fitting (GraphPad Software, San Diego).↵ NyaExecuteng L, et al. (2008) Desorption electrospray ionization reactions between host crown ethers and the influenza neuraminidase inhibitor oseltamivir for the rapid screening of Tamiflu. Analyst 133:1513–1522.LaunchUrlCrossRefPubMed↵ NyaExecuteng L, et al. (2009) Desorption electrospray ionization mass spectrometry (DESI MS) of natural products of a marine alga. Anal Bioanal Chem, in press.
Like (0) or Share (0)