MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Summary

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described. 

Abstract

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied¹. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples^1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

Introduction

The growing field of metabolomics has many important biological applications including biomarker discovery, deciphering metabolic pathways in plants and other biological systems, and toxicology profiling^4,6-10. A major technical challenge when studying biological systems is to study metabolomic pathways without disrupting them¹¹. MALDI-MSI allows for direct analysis of intact tissues that enables sensitive detection of analytes in single organs^12,13 and even single cells^14,15.

Sample preparation is a crucial step in producing reproducible and reliable mass spectral images. The quality of the images greatly depends upon factors such as tissue embedding medium, slice thickness, MALDI matrix, and matrix application technique. For imaging applications, ideal section thickness is the width of one cell (8-20 µm depending on the sample type). MALDI requires deposition of an organic, crystalline matrix compound, typically a weak acid, on the sample to assist analyte ablation and ionization.¹⁶ Different matrices provide different signal intensities, interfering ions, and ionization efficiencies of different classes of compounds.

The matrix application technique also plays a role in the quality of mass spectral images and different techniques are appropriate for different classes of analytes. Three matrix application methods are presented in this protocol: airbrush, automatic sprayer, and sublimation. Airbrush matrix application has been widely used in MALDI imaging. The advantage of airbrush matrix application is that it is relatively fast and easy. However, the quality of the airbrush matrix application greatly depends on the skill of the user and tends to be less reproducible and cause diffusion of analytes, especially small molecules¹⁷. Automatic sprayer systems have similar mechanics to airbrush matrix application, but have been developed to remove the variability seen with manual airbrush application, making the spray more reproducible. This method can sometimes be more time-consuming than traditional airbrush matrix application. Both manual airbrush and automatic sprayer systems are solvent-based matrix application methods. Sublimation is a dry matrix application technique that is becoming more and more popular for mass spectral imaging of metabolites and small molecules because it reduces analyte diffusion; however, it lacks the solvent necessary to extract and observe higher mass compounds¹⁸.

Confident identification of metabolites typically requires accurate mass measurements to obtain putative identifications followed by tandem mass (MS/MS) experiments for validation, with MS/MS spectra being compared to standards, literature, or theoretical spectra. In this protocol high resolution (mass resolving power of 60,000 at m/z 400), liquid chromatography (LC)-MS is coupled to MALDI-MSI to obtain both spatial information and confident identifications of endogenous metabolites, using Medicago truncatula roots and root nodules as the biological system. MS/MS experiments can be performed directly on the tissue with MALDI-MSI or on tissue extracts with LC-MS and used for the validation of metabolite identifications.

This protocol provides a simple method to map endogenous metabolites in M. truncatula, which can be adapted and applied to MSI of small molecules in various tissue types and biological systems.

Protocol

1. Instrumentation

1. MALDI-TOF/TOF MSI. Use a mass spectrometer equipped with a MALDI source for analysis of small molecules (see Table of Materials/Equipment). Perform acquisitions in positive or negative ion mode depending on the analytes of interest. Specify a mass range of interest and collect 500 laser shots/spot at 50 µm intervals in both the x and y dimensions across the surface of the sample to generate ion images. The raster width and number of laser shots can be adjusted to obtain higher spatial resolution and maximum signal intensity respectively. Use DHB matrix peaks or internal standards applied to the slide or directly to the tissue to calibrate the mass spectra.
2. High Resolution LC-MS. (see Table of Materials/Equipment) Run sample extracts with LC-MS using either reversed phase (RP) LC with a C-18 column, or normal phase (NP) with a HILIC column depending on the analytes of interest. Use mobile phases and gradients as appropriate for the specific sample type. Perform acquisitions in positive or negative ion modes depending on the sample type.

2. Tissue Preparation

1. Trim the root nodule from the plant, leaving 3-4 mm of root attached to the nodule.
2. Immediately after dissection, use forceps to place the tissue in a cryostat cup and cover with gelatin (100 mg/ml in deionized water). It is essential for the tissue to be stuck to the bottom of the cup with no air bubbles.
3. Flash freeze the tissue by placing the cup in a dry ice/ethanol bath until the gelatin hardens and becomes opaque. Store samples at -80 °C until use.
4. Remove samples from the -80 °C freezer, cut away the plastic cryostat cup and trim away excess gelatin. Mount the embedded tissue to the cryostat chuck with a dime-sized amount of optimal cutting temperature (OCT) media, while not letting the OCT touch the tissue. Place in cryostat box until the OCT solidifies.
5. Allow the chuck and gelatin to equilibrate in the cryostat box (set to -20 or -25 °C) for approximately 15 min.
6. Use the cryostat (see Table of Materials/Equipment) to section tissue approximately the thickness of one cell (8-20 µm depending on the tissue type) and thaw mount each slice onto the ITO-coated glass by warming the back (non-ITO-coated side) of an ITO-coated glass slide on the back of your hand. Place the ITO-coated side of the warmed slide near the frozen tissue slice and allow the slice to stick onto the slide. Placing the sections close together on the slide will provide better alignment during MSI.

3. Matrix Application

1. Airbrush Application of MALDI Matrix
   1. All airbrush procedures should be performed in a fume hood.
   2. Thoroughly clean the airbrush solution container and nozzle (see Table of Materials/Equipment) with methanol and fill the solution container with DHB matrix solution (150 mg/ml in 50% methanol/0.1% TFA v/v).
   3. Hold the airbrush approximately 35 cm from the sample and apply 10-15 coats of matrix on the surface of the slide with a duration of 10 sec spray and 30 sec drying time in between each coat.
   4. Thoroughly clean the airbrush again with methanol when finished to avoid clogging from the matrix solution.
2. Automatic Sprayer Application of MALDI Matrix
   1. Follow the start-up instructions provided by the manufacturers of the automatic sprayer system.
   2. For MSI of metabolites in root nodules using 40 mg/ml (in 50% methanol/0.1% TFA v/v) DHB as the matrix, set the temperature to ~80 °C, velocity to 1,250 mm/min, flow rate to 50 µl/min, and number of passes to 24. For best coverage, it is recommended to rotate the nozzle 90° and/or offset the nozzle 1.5 mm between each pass. Start sprayer method.
      As a side note, the particular sprayer system used here heats the nozzle for faster evaporation of the solvent. As the solvent evaporates, the concentration of the matrix quickly increases. The matrix applied to the sample with the airbrush and the automatic sprayer have comparable concentrations.
   3. Follow the shut-down instructions provided by the manufacturers of the automatic sprayer system.
3. Sublimation Application of MALDI Matrix
   1. Weigh out 300 mg DHB into the bottom of the sublimation chamber (see Table of Materials/Equipment).
   2. Stick the glass slide to the cold finger (top portion of the sublimation chamber) with the tissue sections facing down with double-sided, conductive tape. Cover the entire back of the slide with the double sided tape for even conductivity, producing even matrix deposition.
   3. Clamp the top and bottom halves of the sublimation chamber together with the C-clamp. Connect the vacuum and add ice and cold water to the top reservoir.
   4. Place sublimation chamber in a heating mantle that is at room temperature.
   5. Turn on the vacuum pump. Wait 15 min and turn on the heating mantle. The heating mantle should reach 120 °C over the course of 10 min.
   6. After 10 min, turn off the heat, close valve to vacuum (so the inside of the chamber remains under vacuum) and turn off the vacuum pump.
   7. Allow the chamber to come to room temperature, open the valve releasing the vacuum pressure and remove the sample. The size of the sublimation chamber will determine the amount of matrix sublimed to the glass slide. The larger sublimation chambers (the size of a 400 ml beaker) will use approximately 300 mg of DHB while the smaller chambers (the size of a 150 ml beaker) will use approximately 100 mg of DHB and will require cutting the glass slide so that it fits in the chamber.

4. Image Acquisition

1. Mark a + pattern on each corner of the sample with a WiteOut correction fluid pen to be used as "teach points". Place the glass slide into the MALDI slide adapter plate and take an optical image of the sample using a scanner.
2. Set up an image acquisition file using the software provided by the instrument company with a raster step size of 50 µm and a laser diameter equal to or smaller than the raster step size. On this particular instrument, the minimum laser setting gives a laser diameter of approximately 10 µm and small laser setting has a 40-50 µm diameter.
3. Load the optical image into the software and align the plate with the optical image.
4. Calibrate the instrument before beginning the acquisition using common matrix cluster ions, internal standards, or a calibration mix.
5. Specify the areas of tissue to be analyzed with MSI, including a spot of pure matrix on the slide to be used as a "blank".
6. Begin acquisition.

5. Image Generation

1. Open the imaging file in the commercially available software provided by the vendor and extract the ion images. Other open-source software is available for MSI data processing¹⁹.

6. Metabolite Identification

1. Select a specific m/z of interest from the mass spectrum using the vendor specific software (see Table of Materials/Equipment). An analyte can be distinguished from a matrix ion when an analyte peak is selected from the mass spectrum and ion images are generated specifically localized to the tissue section.
2. Generate a list of analytes of interest and perform MS/MS experiments. See Table 1 and Table 2 in Representative Results for sample lists of analytes.
3. Perform targeted LC-MS analysis on a high resolution mass spectrometer (or high resolution MALDI-MS if available) to obtain accurate mass measurements of the analytes of interest and also perform LC-MS/MS of the target analytes to obtain characteristic fragmentation patterns.
4. Perform database searching to determine putative identifications for the targeted analytes. Examples of metabolite databases include: METLIN, ChemSpider, PubChem, KEGG, and HMDB.
5. To confirm the putative identifications from accurate mass database searching, match the MS/MS from the targeted analytes to MS/MS spectra of standards, literature, and/or fragmentation prediction software.

Results

An experimental overview of MSI is shown in Figure 1. At the very beginning of the experiment, sample preparation is a critical step. Nodules are trimmed from the plant root and embedded in gelatin. The tissue must be pressed flat against the cryostat cup, with no bubbles, while it is being frozen; this will ensure easier and proper alignment of the tissue while it is being sectioned. When the tissue is being sliced, it is important to cut the tissue at the proper thickness; too thin of sections will tea...

Discussion

As discussed above, sample preparation is the most critical step in the MSI workflow. Embedding the tissue unevenly will cause sectioning to be difficult or not possible in some cases. The section size and adequate equilibration time are crucial to maintaining the tissue integrity and avoiding folding and tears. Selection of matrix and application technique will play a role in determining the types of analytes to be detected, the spatial resolution, and reproducibility of the results. Using a combination of matrices or a...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gelatin	Difco	214340	heat to dissolve
Cryostat- HM 550	Thermo Scientific	956564A
Indium tin oxide (ITO)-coated glass slides	Delta Technologies	CB-90IN-S107	25 mm x 75 mm x 0.8 mm (width x length x thickness)
2,5-Dihydroxybenzoic acid (DHB) matrix	ICN Biomedicals	PI90033
Airbrush	Paasche Airbrush Company	TG-100D	coupled with 75 ml steel container
Automatic matrix sprayer system- TM-Sprayer	HTX Technologies, LLC	HTX.TMSP.H021-U	Specific start-up and shut-down instructions will be given when the instrument is installed
Sublimation apparatus	Chemglass Life Science	CG-3038-01
Vaccum pump- Alcatel 2008 A	Ideal Vacuum Products	P10976	Ultimate Pressure = 1 x 10-4 Torr
ultrafleXtreme MALDI-TOF/TOF	Bruker Daltonics	276601
FlexImaging	Bruker Daltonics	269841	One example of "vender specific software"
MALDI LTQ Orbitrap	Thermo Scientific	IQLAAEGAAPFADBMASZ	High resolution MALDI instrument for accurate mass measurements
Q Exactive	Thermo Scientific	IQLAAEGAAPFALGMAZR	High resolution LC-MS instrument for accurate mass measurements