Application Note #44 | 07/2018

Application & Background Visualizing microbial interactions with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has provided many insights into important biological processes, including metabolic exchange, antibiotic resistance, and microbial competition.1,2 As the application of MALDI-MSI technology to microbial science continues to grow, it is important that validated protocols are shared and established in the literature. Here, we detail a complete experimental workflow for MALDI-MSI of microbial cultures grown on agar, wherein we investigate the different ecological roles of members of a peatland environment. Peatland ecosystems occupy 3% of the Earth’s land surface yet store 25-33% of terrestrial carbon as recalcitrant organic matter. In the peatland of Marcell experimental forest in northern Minnesota, the ecosystem is dominated by the moss (Sphagnum fallax ), which associates with nitrogen-fixing cyanobacteria (Nostoc muscorum ) and the fungus (Trizodia spp. ), but these interactions are not well characterized. In this study, under the mission of the Spruce-Peatland Response Under Climate and Environmental Change (SPRUCE) Department of Energy (DOE), we aimed to identify the role of various peatland species and to investigate the metabolic communication between them in order to better understand and control biodiversity within this complex ecosystem.3

ExperimentalSample PreparationFor tripartite interactions, each organism was positioned approximately 2 cm from one another and for individual control, a single organism was placed in the center of the plate.2 All samples to be analyzed by MALDI IMS were grown on 1.5 mm thick BG-110 (pH 5.5) 1.5% agar plates at 24°C with a 16 hr/8 hr (day/night) cycle at 150 PAR. The petri dishes were sealed with Parafilm to minimize dehydration of thin agar during incubation and incubated for 12 days. After the incubation period, agar areas of the tripartite interaction and of isolated culture controls were excised from petri dishes and were carefully placed onto an indium tin oxide (ITO)-coated glass slide using a spatula. Since the moss excessively protruded from the surface of the agar, plant tissue was carefully removed prior to dehydration to assist in IMS analyses. Samples were then dried under ambient conditions in a BSL hood overnight.2

Mass Spectrometry Imaging of Moss, Fungus, and Cyanobacteria on Agar to Investigate Tripartite Metabolic Interactions

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APPLICATION NOTE

Figure 2. (A) The tripartitre interation of the moss (Sphagnum fallax), nitrogen-fixing bacteria (Nostoc muscorum), and the fungus (Trizodia spp.) grown in a petri dish on 1.5% BG110 agar. (B) The agar area of the tripartite interaction was excised from the petri dish and transferred onto an ITO coated glass slide. Samples were dehydrated overnight at ambient temperature and ambient pressure before DHB MALDI matrix was applied using the HTX TM-Sprayer.

Figure 1. The HTX TM-Sprayer for MALDI matrix deposition.

(A)

(B)

References1Anderton, C. R., Chu, R. K.,Tolic, N., Creissen, A. and Pasa-Tolic, L. (2016). "Utilizing a Robotic Sprayer for Hig Lateral and Mass Resolution MALDI FT-ICR MSI of Microbial Cultures." Journal of the American Society for Mass Spectrometry 27(3): 556-559.2Velickovic, D., Chu, R.K., Carrell, A.A., Thomas, M., Pasa-Tolic, L., Weston, D. J. and Anderton, C. R. (2018). "Multimodal MSI in Conjunction with Broad Coverage Spatially Resolved MS2 Increases Confidence in Both Molecular Identification and Localization." Analytical Chemistry 90(1): 702-707.3Hanson, P. J., Childs, K. W., Wullschleger, S. D., Riggs, J. S., Thomas, W.K., Todd , D. E. and Warren, J. M. (2011). "A method for experimental heating of intact soil profiles for application to climate change experiments." Global Change Biology 17(2): 1083-1096.).4Privalle, L. S. and Burris, R. H. (1983). "Adenine-Nucleotide Levels in and Nitrogen-Fixation by the Cyanobacterium Anabaena Sp Strain-7120." Journal of Bacteriology 154(1): 351-355.5Laparre, J., Malbreil, M., Letisse, F., Portais, J. C., Roux, C., Becard, G., and Puech-Pages, V. (2014). "Combining Metabolomics and Gene Expression Analysis Reveals that Propionyl- and Butyryl-Carnitines Are Involved in Late Stages of Arbuscular Mycorrhizal Symbiosis." Molecular Plant 7(3): 554-566.6Rivoal, J. and A. D. Hanson (1994). "Choline-O-Sulfate Biosynthesis in Plants - Identification and Partial Characterization of a Salinity-Inducible Choline Sulfotransferase from Species of Limonium (Plumbaginaceae)." Plant Physiology 106(3): 1187-1193.7Franken, J., Burger, A., Swiegers, J. H., and Bauer, F. F. (2015). "Reconstruction of the carnitine biosynthesis pathway from Neurospora crassa in the yeast Saccharomyces cerevisiae." Applied Microbiology and Biotechnology 99(15): 6377-6389. 8Wang, M. L., Moynie, L.,Harrison, P. J., Kelly, V., Piper, A., Naismith, J. H. and Campopiano, D. J. (2017). "Using the pimeloyl-CoA synthetase adenylation fold to synthesize fatty acid thioesters." Nature Chemical Biology 13(6): 660-+.

Results Using spatial segmentation image analysis in SCiLS, 288 ion images associate with members of tripartite community (Figure 4). For 213 ion images, molecular formulas are assigned based on high accurate m/z. These data are also uploaded in METASPACE, and are publically available for browsing here.*

As seen in Figure 5, adenine was found to co-localize with the cyanobacteria. This was an expected finding as adenine is known to play an important role in maintaining energy balance during the nitrogen fixation process.4 Butyryl-carnitine, described as a signaling molecule in plant-fungus symbiosis,5 was almost exclusively mapped around the fungi. Finally, choline-sulfate, a plant osmoprotectant,6

showed co-localization with the plant part of the tripartite community.

APPLICATION NOTE

Flow Rate (FR) 0.100 mL/min

Spray Nozzle Velocity (V) 1200 mm/min

Spray Nozzle Temperature 65º C

Track Spacing (TS) 3 mm

Number of Passes (NP) 12

Matrix density (W) W = NP ×C ×FR V ×TS

= 0.013 mg/mm2

Linear Flow Rate (LFR) LFR = FRV = 8.3e-5 mL/min*

Matrix ApplicationDHB was applied to the slides at a concentration (C) of 40 mg/mL (in 70:30 methanol:water) using the HTX TM-Sprayer Sprayer. The slides were coated using the following parameters:

* Corresponds to dry spray, defined by LFR between 8.3e-5 and 8.3e-6

MALDI Imaging Mass SpectrometryImaging was performed on a 15 Tesla MALDI FTICR-MS (SolariX, Bruker Daltonics) equipped with a SmartBeam II laser source (355 nm, 2 kHz) in positive mode, using 200 shots at 2kHz and a 200 µm step size. The SolariX FTICR-MS was operated to collect m/z 92-1000, using a 209 ms transient, which translated to a mass resolution of R ~ 130,000 at m/z 400. Data was acquired using FlexImaging (v4.1, Bruker Daltonics), and imaging processing and visualization were performed using SCiLS Lab. Figure 4. SCiLS spectral segmentation of tripartite interaction

Figure 5. Distribution of some metabolites involved in tripartite communication. Lateral resolution: 200 µm

Nitrogen Pressure 10 psi

Figure 3. 15 Tesla Bruker SolariX MALDI FTICR-MS equipped with SmartBeam II laser source

*http://metaspace2020.eu/#/annotations?ds=2018-06-14_02h49m16s&fdr=0.5&q=velickovic&sort=-msm&cmap=Cividis&sections=15

Application Note #44 | 07/2018

Experimental Summary Tissue Type

Incubation Protocol

Agar Plates

Moss (S. fallax), cyanobacteria (N. muscorum), and fungus (T. spp.)

1.5% Agar plates, 1.5 mm thick BG-110 (pH 5.5)

12 days at at 24°C with a 16 hr/8 hr (day/night) cycle at 150 PAR

MALDI Plate Matrix

Matrix Sprayer MALDI MS Laser SourceAcquistion ModeImaging Software

Instrumentation and Supplies ITO coated glass slidesDHB (Sigma Aldrich, 40 mg/mL in 70:30 MeOH/H2O) HTX TM-Sprayer™Bruker SolariX 15T FT-ICR Smartbeam II, Positive Mode Fullscan (m/z 92-1000) FlexImaging v4.1 SCiLS Lab

Figure 6. Tracking the metabolic routes of possible candidates of ion m/z 189.1598 in spatial manner in order to identify which of two natural compounds it represents. Co-localization of ion m/z 266.0934 and m/z 189.1598, and absence of any co-localization between m/z 162.1125 and m/z 189.1598 indicate that structure of ion with molecular formula C9H20N2O2 is as in 7,8 diaminononaoate. Lateral resolution: 200 µm.

Conclusions

In MALDI IMS experiments involving complex systems composed from several species, tracking spatial metabolism of specific compounds can be an efficient solution for the structural identification of these compounds. In this study, the ion m/z 189.1598 was identified as [C9H20N2O2 + H]+ and was distributed around the fungus in the tripartite interaction. This ion can be ascribed to two previously identified natural compounds: 7,8-diaminononanoate and trimethyllysine, metabolites with related, but distinct biological roles in fatty acid metabolism (Figure 6). Specifically, trimethyllysine is precursor of carnitine biosynthesis, a molecule that serves as a fatty acid shuttle during lipid catabolism,7 while 7,8-diaminononanoate plays a role in the biosynthesis of biotin, an essential carbon dioxide carrier cofactor for fatty acid synthesis.8

When we imaged the end-products of these 2 biosynthetic routes we can see much higher co-localization (Pearson correlation coefficient 0.7) of biotin amide than carnitine (Pearson correlation coefficient 0.0) with [C9H20N2O2 + H]+ ion. This indicate that observed ion is rather 7,8-diaminonanoate than trimethyllisine. Note that MS/MS data are not able to unambiguously resolve these two isomers,2 so spatial metabolomics could be advanced approach in such cases.

The tissue images and MS data presented in this note were provided by Dr. Dusan Velickovic and Dr. Christopher Anderton from Pacific Northwest National Laboratory (PNNL).

HTX M5 Sprayer™ is available worldwide exclusively from HTX Technologies, LLC.

To request further information contact:

Alain Creissen Imaging Product Manager, HTX Technologies

acreissen@htximaging.com

HTX Technologies offers innovative sample preparation systems for advanced analytical platforms. Our integrated workflow solutions include user training, instruments, software, consumables and method development services.

PO Box 16007 Chapel Hill, NC 27516, USA

Tel +1-919-928-5688 Fax +1-919-928-5154

info@htximaging.com www.htximaging.com

HTX M5 Sprayer™ System is an

Automated MALDI Matrix Deposition System

Offering High Reproducibility and Superior

Data Quality for Imaging Mass Spectrometry

The HTX M5 Sprayer™ is an easy-to-use, versatile spraying system that provides automated processes for sample preparation in imaging mass spectrometry.

The proprietary spray technology of the HTX M5 Sprayer™ guarantees a very fine, uniform and consistent matrix coating crucial for high-resolution imaging and relative quantification of analytes.

The unique ability to control liquid and propulsion gas temperature creates a fine solution mist that can be deposited in a precise and adjustable pattern over all or part of any MALDI plate.

Spray characteristics (wet or dry) are easily adjustable via the intuitive operator interface. Users can create and save methods for reproducible operation.

Key Characteristics◆ Proprietary technology providing very small matrix

droplets ( <5 microns)

◆ High flow rate and fast sample prep (2 to 18 minutesper slide)

◆ Highly consistent matrix deposition across entiresample area (+/- 3% by weight)

◆ Unique use of temperature and nitrogen flow tocontrol evaporation rate and matrix crystal formation

◆ More than 30 validated protocols covering trypsinand most matrices (e.g.: SA, CHCA, DHB, DAN,9-AA, DHA, CMBT, THAP)

◆ Validated protocols for Trypsin digestion of FFPE

◆ Continuous matrix coverage as needed forhigh-resolution imaging

◆ Rugged operation and easy clean-up

Addressing the Matrix Deposition Challenge The main challenge when preparing samples for MALDI Mass Spectrometry Imaging is to balance the positive effects of the matrix solution penetrating the tissue and co-crystallizing with the analyte, and the negative effects of analytes delocalization.

The all-new M5 chassis, high velocity stage and heated sample holder drawer contribute to a greater user experience and expanded process capabilities including:

◆ Faster and drier deposition capability

◆ On-tray trypsin digestion capability

◆ On-tray sample re-hydration

HTX M5 Sprayer™ Tissue MALDI Sample Preparation System TECHNICAL NOTE

Technical Note #28 | 052013 | © 2014 HTX Technologies.

Application2,5-dihydroxybenzoic acid (DHB) is a matrix suitable for imaging metabolites. DHB can easily be dissolved in 50:50 Water:Methanol (0.1% Trifl uoroacetic Acid) solution. When imaging metabolites DHB can be sprayed directly onto tissue sections with no pre-treatment, but may show interfering peaks in the low mass region of the mass spectra. Th e data presented here were obtained as part of a MALDI MS imaging experiment whose purpose was to detect metabolites present in the root nodules of the Medicago truncatula – Sinorhizobium meliloti symbiosis during nitrogen fi xation.

Intended Use Of This Technical NoteTh e information provided in this document was not peer reviewed and is intended to illustrate possible uses of the TM-Sprayer. HTX Technologies, the manufacturers referenced in this note and the users that have accepted to share their data do not make any guarantees as to the performance of the illustrated workfl ow.

Imaging Workfl owRoot nodules were dissected out of the plant, embedded in gelatin (100 mg/ml in deionized water), and frozen on dry ice. Cryosections (14 microns) of snap frozen root nodule tissue was mounted on ITO-coated glass slides.

Tissue sections were then sprayed with DHB matrix (40mg/ml, Methanol 50%, TFA 0.1%) using the HTX TM-Sprayer and the following conditions:

Flow Rate 50 µl/min

Spray Nozzle Velocity 1250 mm/min

Spray Nozzle Temperature 80°C

Track Spacing 3 mm

Number of Passes 24, criss-cross and offset

Nitrogen Pressure 10 psi

Optimization of DHB Matrix Spray for MALDI Imaging of Metabolites in Root Nodule Tissue of the Medicago truncatula – Sinorhizobium meliloti Symbiosis

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TECHNICAL NOTE

Technical Note #28 | 052013 | © 2013 HTX Technologies.

Application2,5-dihydroxybenzoic acid (DHB) is a matrix suitable for imaging metabolites. DHB can easily be dissolved in 50:50 Water:Methanol (0.1% Trifluoroacetic Acid) solution. When imaging metabolites DHB can be sprayed directly onto tissue sections with no pre-treatment, but may show interfering peaks in the low mass region of the mass spectra. The data presented here were obtained as part of a MALDI MS imaging experiment whose purpose was to detect metabolites present in the root nodules of the Medicago truncatula – Sinorhizobium meliloti symbiosis during nitrogen fixation.

Intended Use Of This Technical NoteThe information provided in this document was not peer reviewed and is intended to illustrate possible uses of the TM-Sprayer. HTX Technologies, the manufacturers referenced in this note and the users that have accepted to share their data do not make any guarantees as to the performance of the illustrated workflow.

Imaging WorkflowRoot nodules were dissected out of the plant, embedded in gelatin (100 mg/ml in deionized water), and frozen on dry ice. Cryosections (14 microns) of snap frozen root nodule tissue was mounted on ITO-coated glass slides.

Tissue sections were then sprayed with DHB matrix (40mg/ml, Methanol 50%, TFA 0.1%) using the HTX TM-Sprayer and the following conditions:

Flow Rate 50 µl/min

Spray Nozzle Velocity 1250 mm/min

Spray Nozzle Temperature 80°C

Track Spacing 3 mm

Number of Passes 24, criss-cross and offset

Nitrogen Pressure 10 psi

Optimization of DHB Matrix Spray for MALDI Imaging of Metabolites in Root Nodule Tissue of the Medicago truncatula – Sinorhizobium meliloti Symbiosis

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Spectra were collected across the entire tissue area usingthe ultrafleXtreme MALDI-TOF/TOF (Bruker Daltonics, Billerica, MA, USA) analyzer equipped with a 2 kHz, FlatTop smartbeam-II™ Nd:YAG laser in reflectron positive mode over a mass range of m/z 80 to 1000. A total of 500 laser shots were accumulated and averaged from each laser spot, using the “minimum” laser spot diameter setting and a raster width of 50µm. Calibration was performed externally using DHB cluster peaks in the mass range of m/z 100 to 750.

Figure 1. Methylene blue stained Medicago truncatula root nodule(Image courtesy of Dr. Jean-Michel Ané lab in the Department of Agronomy at UW-Madison.)

TechNote28.indd 1 6/4/13 8:43 PM

Spectra were collected across the entire tissue area usingthe ultrafl eXtreme MALDI-TOF/TOF (Bruker Daltonics, Billerica, MA, USA) analyzer equipped with a 2 kHz, FlatTop smartbeam-II™ Nd:YAG laser in refl ectron positive mode over a mass range of m/z 80 to 1000. A total of 500 laser shots were accumulated and averaged from each laser spot, using the “minimum” laser spot diameter setting and a raster width of 50µm. Calibration was performed externally using DHB cluster peaks in the mass range of m/z 100 to 750.

Figure 1. Methylene blue stained Medicago truncatula root nodule(Image courtesy of Dr. Jean-Michel Ané lab in the Department of Agronomy at UW-Madison.)

The HTX TM-Sprayer™

System is an automated

MALDI matrix deposition

system offering

high reproducibility and

superior data quality for

Mass Spectrometry Imaging

Th e HTX TM-Sprayer™ is an easy-to-use, versatile spraying system that provides an automated process for Sample Preparation in Mass Spectrometry Imaging.

Th e patented spray technology of the TM-Sprayer™ guar-antees a very fi ne, uniform and consistent matrix coating crucial for high-resolution imaging and relative quantifi ca-tion of analytes.

Th e new HTX Technologies’ spray nozzle, featured in the next generation TM-Sprayer, creates a fi ne solvent mist that can be deposited in a precise and adjustable pattern over all or part of any MALDI plate.

Spray characteristics (wet or dry) are easily adjustable via the intuitive operator interface. Users can create and save methods for reproducible operation.

Key Characteristics Patented technology providing very small matrix droplets

( <10 microns) High fl ow rate and fast sample prep (10 to 20 minutes

per plate )

Highly consistent matrix deposition across entire samplearea (+/- 3% by weight)

Unique use of temperature and nitrogen fl ow to controlevaporation rate and matrix crystal formation

Validated protocols for most matrices(e.g.: SA, CHCA, DHB)

Validated protocols for Trypsin digestion

Continuous matrix coverage as needed forhigh-resolution imaging

Rugged operation and easy clean-up

TM-Sprayer™ Specifi cationsDeposition: Spray deposition in linear or serpentine modes with variables off setsSpray Nozzle Flow: 50 to 1000µl/minSheath Gas: Ambient to 130°C (+/- 2°C), software selectedGas Supply: Sheath gas fl ow 5-15.5 liter/minSpray Nozzle Position: Spray nozzle mounted on Cartesian stage Electrical: 24V Power SupplyDimensions/Weight: 17 x 15 x 13in (43 x 38 x 33cm),38lbs (17Kg)

HTX Technologies, LLC offersinnovative sample preparation systems for advanced analytical platforms. For more information please contact ERC GmbH or visit www.htxtechnologies.com

ERC GmbHOtto-Hahn-Str. 28-3085521 RiemerlingGERMANY

Tel. +49 89 66055696Fax +49 89 60824826partner@erc-hplc.dewww.erc-hplc.de

TM-Sprayer™ Tissue MALDI Sample Preparation System

TechNote28_ERC.indd 1-2 12/30/14 10:07 AM