Direct Protocol for Ambient Mass Spectrometry Imaging on Agar Culture

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A direct protocol for ambient mass spectrometry imaging on agar culturere Célio Fernando Figueiredo Angolini, Pedro Henrique Vendramini, Francisca Diana da Silva Araujo, Welington L. Araújo, Rodinei Augusti, Marcos Nogueira Eberlin, and Luciana Gonzaga de Oliveira Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 11 Jun 2015 Downloaded from http://pubs.acs.org on June 12, 2015

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Analytical Chemistry

A direct protocol for ambient mass spectrometry imaging on agar culture Célio Fernando F. Angolinia,*, Pedro Henrique Vendraminia, Francisca D. S. Araújoa, Welington L. Araújob, Rodinei Augustic, Marcos N. Eberlina, Luciana Gonzaga de Oliveiraa,* a

Department of Organic Chemistry, Institute of Chemistry, University of Campinas, UNICAMP 13083-970 Campinas – SP, Brazil

b

Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, 05508900 São Paulo – SP, Brazil c

Department of Chemistry, Federal University of Minas Gerais - UFMG, 31270-901, Belo Horizonte, MG, Brazil.

Abstract: Herein we describe a new protocol that allows direct mass spectrometry imaging (IMS) of agar cultures. A simple sample dehydration leads to a thin solid agar, which enables the direct use of spray-based ambient mass spectrometry techniques. To demonstrate its applicability, metal scavengers siderophores were imaged directly from agar culture of S. wadayamensis and well resolved and intense images were obtained using both desorption electrospray ionization (DESI) and easy ambient sonic-spray ionization (EASI) with well-defined selective spatial distributions for the free and the metal-bound molecules, providing clues for their roles in cellular metabolism.

Streptomyces are Gram-positive bacteria with a unique capacity to produce metabolites with roles ranging from normal physiological processes in the primary metabolism to complex ecological functions in secondary metabolism.1 The functional variety is reflected in the structural diversity of these metabolites, ranging from small molecules and iron scavengers to large peptides and proteins. Because more than 50% of all therapeutic drugs originate from natural products, the importance of detecting and characterizing such molecules with both time and space resolution and the need for novel methodologies to perform such screening has been and remains high. Individual metabolites have traditionally been targeted using demanding and timeconsuming bioactivity-guided fractionation from the crude microbial extracts. These bioactive molecules have now begun to be selectively monitored with different time and spatial distributions using mass spectrometry (MS) techniques, exponentially expanding the frontiers of microbial metabolomics.2,3 Imaging mass spectrometry (IMS) has emerged as a powerful tool for surface screening at the molecular level and different approaches are used.2-5 “Invacuum” and ambient ionization have been constantly used for the imaging of microbial bioactive molecules.6-8 Some “in-vacuum” techniques allow for the direct analysis of agar culture, such as those using matrix-assisted laser desorption ionization (MALDI) and secondary ion mass spectrometry (SIMS), however they can be time-consuming for data acquisition and/or sample preparation.3,9 For ambient ionization, analysis of agar culture were also demonstrated using laser desorption post ionization (LDPI)8 and desorption electrospray ionization (DESI). Agar culture analysis using DESI-MS was mostly accomplished via an imprinting process, where the bias of the imprinting leads to loss of information on molecular spatial distribution.2 A recently alternative, namely nanoDESI,10,11 has brought new possibilities

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on MS imaging of living microbial colonies directly from the agar, but frequent adjustments need to be done during image acquisition for proper droplet-surface contact. IMS on agar surfaces has been therefore a challenge task, and direct ambient MS analysis has been only sporadically tried. Herein, we describe a new, fast (when compared to MALDI-IMS), and more informative protocol (as compared to the imprinting process) for the MS imaging of metabolites directly from the surface of dehydrated agar cultures. For detection, we mostly used DESI-IMS but EASI-IMS is demonstrated to produce images of similar high quality. As a test case, the spatial distribution of metabolites involved in the iron uptake mechanism of an endophytic microorganism from Citrus reticulate, identified as Streptomyces wadayamensis strain A23, was screened.12

EXPERIMENTAL SECTION Chemicals and samples. Water was purified using a Milli-Q water purification system (Millipore). Beef extract, yeast extract, peptone and agar were purchased from Oxoid (Hampshire, UK). HPLC-grade methanol was purchased from TediaBrasil (Rio de Janeiro, Brazil). FeCl3.6H2O, HCl, chromeazurol S, hexadecyltrimethylammonium bromide (HTMS), PIPES, KH2PO4, NaCl, NH4Cl, KOH, glucose, D-mannitol, casein hydrolysate, MgSO4.7H2O, CaCl2, MnSO4.H2O, H3BO3, CuSO4.5H2O, ZnSO4.7H2O and Na2MoO4.2H2O were purchased from SigmaAldrich. The actinobacterium strain A23 identified as Streptomyces wadayamensis was isolated from a population existing in the plant tissue of Citrus reticulata (tangerine). Streptomyces culture using thin-layered agar. For microbial IMS sample preparation, a 0.25 µl sample from a Streptomyces spore solution was inoculated on thin-layered agar. The agar was prepared by first placing a sterile microscope slides in a Petri dish, followed by the pouring of agar medium. Typically, to achieve an ideal thin-layered agar plate, 9-10 mL of medium was needed for a standard 10-cm Petri dish, which results in a 1-1.5 mm agar height. We used antibiotic assay medium 2 (1.5 g L-1 beef extract, 3.0 g L-1 yeast extract, 6 g L-1 peptone and 1.5% agar) however, other media were also tested. The inoculated medium was incubated at 30 ⁰C, and the Petri dish was sealed with parafilm to minimize premature dehydration. DESI sample preparation. After the incubation period, the microscope slides were removed from the Petri dish, photographed and put in a vacuum desiccator for complete agar dehydration at room temperature. The time of desiccation varies, but a maximum of 40 min were needed. For the imprint analyses we followed a reported procedure2 using a PTFE membrane with a 0.22 µm pore size (Allcrom). The same samples were desiccated after imprinting for data comparison. Data Analysis. The DESI-IMS data was converted into imaged files using Firefly data conversion software (version 2.1.05) and viewed using the BioMAP software (version 3.8.04). In BioMAP the data were opened in an m/z 500-700 range and the false-color scaling were adjusted to a fixed value to enable a relative comparison between experiments. Instrumentation. MS imaging was performed using a Prosolia DESI source (Model OS-3201) coupled to a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer. The DESI configuration was set with an emitter height of 2.5 mm, mass spectrometer inlet height of 0.1 mm, inlet to emitter distance of 3.8 mm, 58⁰ spray angle, 5.0 kV spray voltage, inlet capillary temperature of 320 ⁰C, 100 V S-lens, 160 psi ultra-pure nitrogen nebulizing gas


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pressure and a sprayed solvent of methanol at a 3.0 µL min-1 flow rate. Images were collected from m/z 133-1500 with a step sized of 200 µm, a scan rate of 741 µm sec-1 and a pixel size of 200 µm x 200 µm. For EASI-MS imaging, an optimized prototype source mounted as described formerly4 was used. In short, major conditions were: 3 µL min-1 of solvent flow rate, nebulizing gas backpressure of 100 psi and 2 mL min-1 gas flow rate.

RESULTS AND DISCUSSIONS Sample preparations challenges. To maintain a consistent signal for DESI, its desorption momentum-transfer-based mechanism ideally requires a hard, uniform and nonconductive sampling surface.13 As noted by Watrous et al.,2 these requirements had thus far prevented the direct analysis of metabolites from microorganism culture on nutrient agar by DESI-MS imaging, and imprinting had to be employed with the resulting loss of spatial molecular information. We therefore envision strategies that would optimize the microorganism culture and sample preparation protocols to allow the direct analysis of agar cultures by DESI-IMS. We also noticed that during development in agar plate culture, certain microorganisms, especially actinomycetes, could show diverse morphological behaviors. Sometimes growth beyond the surface of the agar is noted, turning the agar surface irregular and making DESI-IMS impracticable. To overcome this limitation, we grew these microorganisms in a thin-layered agar plate, where the reduced volume of agar avoided such undesirable behavior for up to six days. Other restrictions remained to be solved for the proper MS imaging of agar surfaces. The direct analysis of an agar culture by spray-based techniques such as DESI would shatter the agar away; also the conductive nature of the agar results in drastic signal reduction.2,5,14 Gu et al. reported 15 that the electrical conductivity of agarose gel increases with increasing water volume fraction, consequently we performed agar medium dehydration so that the conductivity was minimized or totally cancelled. This simple procedure leads to a thin, solid dehydrated agar, with a hard, uniform and non-conducting surface, therefore solving the two major restrictions for the direct analysis on agar plates using spray-based ambient MS. Figure 1 shows that, indeed, very clear and well-resolved images can be obtained directly from the dehydrated agar surfaces that permits the clear visualization of different spatial distributions over the colony and/or over the growth media. The direct uses of DESI and EASI also reduces the data acquisition time, since the same area can be analyzed four times faster. Usually an image was collected for an area of 4 cm2 with 200 µm resolution in about 4550 min using DESI-MS whereas for MALDI-MS 3-4 h were required. These spatial distributions of the production of specific metabolites are certainly correlated to different physiological functions and/or different morphological modifications during the development of the S. wadayamensis.



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Figure 1- IMS with different spatial distributions of molecules over the S. wadayamensis culture and agar medium of the same sample; in positive mode: (A) m/z 256, (B) m/z 401 and (C) m/z 1067; and in negative mode: (D) m/z 547, (E) m/z 561 and (F) m/z 946; after a incubation period of 48 h.

From a single DESI-IMS screening on S. wadayamensis, many ions with different spatial distributions were detected, ranging from m/z 140 to 1200. Some of these ions could be identified as antimycin isoforms, cyclic and acyclic ferrioxamines corroborating with the recently sequenced and annotated genome, which shows the strain capability to produce numerous bioactive molecules.12 A comparison with the imprinting process was also done (Figure 2). Note that much images with no loss of spatial molecular information is obtained directly from the dehydrated agar, whereas a non-consistent spatial distribution is clearly promoted by the imprinting process.

Figure 2 – (+) DESI-IMS showing different molecules of the same sample of S. wadayamensis culture obtained by the imprinting (top) and direct method (bottom) analyzes, after a incubation period of 60 h. The imprint optical image was darkened to better imprint visualization.

Siderophores as iron scavengers. In Figure 3, the effectiveness of the new sample preparation protocol was tested via the direct analysis of the S. wadayamensis on agar following six days of growth. Among the detected molecules, four show a spatial distribution pattern compatible


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with the expectations based on Chromeazurol S (CAS)-agar medium assays,16 a halo surrounding the colony that becomes larger with incubation time, which indicate production of iron scavenger molecules (Figures 3 and 4). Three of these ions were characterized as siderophores and were identified as protonated desferrioxamine B ([DFO + H]+, m/z 561), ferrioxamine B complex ([DFO - 2H + Fe]+, m/z 614) and Al-desferrioxamine B complex ([DFO 2H + Al]+, m/z 585) based on their tandem MS fragmentation patterns and comparisons with the literature.17

Figure 3 – Siderophore spatial and temporal distribution visualization by DESI(+)-IMS. (A) Direct agar IMS of S. wadayamensis strain A23; (B) ESI(+)-MS/MS showing the fragmentation patterns of protonated desferrioxamine B (m/z 561) and ferrioxamine B complex (m/z 614).

Siderophores have many functions,18-23 but in microorganisms their principal physiological role is related to metal uptake.24 Microorganisms requires a minimum effective concentration of iron which in excess, however, becomes cytotoxic.25 Therefore, iron bioavailability must to be finely adjusted by the cell.26 Microorganisms can overcome this iron nutritional limitation in the host by retrieving extracellular iron through two general mechanisms: iron acquisition by cognate receptors using siderophores and receptor-mediated iron acquisition from host proteins.24 The mechanisms involved in iron uptake may differ in Gram-positive and Gram-negative bacteria due to differences in the cell membranes and walls.24 For Gram-positive bacteria, two mechanisms are reported: the siderophore shuttle and the siderophore displacement mechanisms.25,27 Both involve a siderophore-binding protein to sequestrate the Apo-siderophore/Fe-siderophore; and a siderophore-pemease(s)-ATPase



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system for Fe-siderophore complex delivery into the cytoplasm. If DESI(+)-IMS could provide the spatial distributions of ferrioxamine B (Fe-siderophore, m/z 614) and desferrioxamine B (Apo-siderophore, m/z 561) throughout the incubation period, important information about the iron uptake mechanism would be obtained. As shown by Figure 3, this information was indeed obtained. An interesting feature of the ion images was the greater intensity of Aposiderophore (m/z 561) over the cell culture in relation to Fe-Siderophore (m/z 614). This trend agreed with Fukushima et al.,27 who showed that increases in the local concentration of the apo-siderophores (m/z 561) facilitate the siderophore shuttle mechanism in Gram-positive bacteria. Actually, DESI(+)-IMS shows that the Fe-siderophore complex (m/z 614) is practically absent throughout the cell culture (Figure 3), which would be correlated to the high efficiency of the iron uptake system of the microorganism. Other experiments should however be performed to confirm this observation, since variation on surface due the presence of microorganism cells can interfere into ionization. Additionally, during microorganism growth, DESI(+)-IMS revealed an increasing halo around the cells with a lower intensity of Aposiderophore (m/z 614) (Figure 3, FO with 96, 120 and 144 h), most likely due to iron depletion by the microorganism.

Siderophores as aluminum bioavailability reducers. In addition to iron siderophore uptake, DESI(+)-MS imaging was able to show a high affinity of desferrioxamine B for aluminun chelation as the ion [DFO - 2H + Al]+ of m/z 585 was also detected in higher abundance than [DFO - 2H + Fe]+. Althought aluminum is the most abundant metal in the Earth´s crust, it lacks biological functions and is considered a nonessential metal.28 However, aluminum is toxic to cells, so it seems possible that desferrioxamine B may be acting in mechanisms of metal toxicity control, with Al-complexation hindering its entrance into the cell membrane.29 It is importante to note that similar observations were recently reported by Moree et al for fusarinine-derived siderophores.30 As Figure 4 indicates, the spatial distribution of [DFO - 2H + Al]+ of m/z 585 reveals Al distribution over the agar media and the bacteria cells. The [DFO - 2H + Al]+ abundance also seems to increase at longer incubation periods, showing that the cell is unable to transport this complex into the cytoplasm.


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Figure 4 – Al-siderophore spatial and temporal distribution visualization by DESI(+)-MS imaging. (A) Direct agar IMS of S. wadayamensis strain. (B) MS/MS fragmentation patterns of the [M - 2H + Al]+ complex (Al-desferrioxamine B), m/z 585.

Easy ambient sonic-spray ionization (EASI)4 is another popular spray-based ambient MS technique.31 Due to the considerably high pressure and flow to produce the spray in EASI, it was important to test the robustness of the dehydrated agar surface to the more aggressive bombardment of EASI droplets for proper IMS. As Figure 5 shows, images of similar quality and same resolution (200 µm) to the ones acquired by DESI-MS were obtained.

Figure 5 - Direct agar EASI-IMS of S. wadayamensis strain A23, 36 h incubation period. (A) m/z 561, (B) m/z 585, (C) m/z 614.

CONCLUSIONS A new protocol for rapid, direct and efficient ambient IMS using spray-based techniques for agar cultures has been demonstrated. The use of a dehydrated agar eliminates most of the limitations previously reported for DESI-MS on hydrated agar surfaces. Agar spray shattering is



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minimized or practically eliminated, and the loss of molecular information observed when based imprinting methods are used is also avoided. Well-resolved and intense images were obtained showing the spatial distributions of metabolites, including the extracellular metabolites that surrounds the microorganism colony and are embedded in the growth media, making the protocol quite advantageous. Another advantage of this method is the resilience of dehydrated agar allied to the spray-based techniques such as DESI and EASI, allowing data to be acquired in both the negative and positive ion modes as well as MS/MS data on target biomarkers from the same sample. As a test case, the method has provided detailed temporal and spatial metabolite monitoring for Streptomyces wadayamensis. A number of key metabolites could be monitored, including the free and the chelated siderophores with both iron and aluminum. This method of direct analysis of bacterial colonies on the agar surface expands the boundaries of spatial imaging mass spectrometry for the monitoring of microorganisms, and brings an additional tool for microbial IMS screening.

ACKNOWLEDGMENTS We are thankful to Professor Anita J. Marsaioli for generously allowing the use of their laboratory to perform the bacterial experiments. Funding was provided by São Paulo Research Foundation (FAPESP, grant 2010/51677-2), PETROBRAS (grant 4712-0), CNPq and IFS (Grant Support No. F/4735-1). LG de Oliveira is also grateful for the grant provided by the program For Women in Science (2008, Brazilian Edition).

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Direct Protocol for Ambient Mass Spectrometry Imaging on Agar Culture

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