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 image....

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....

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