Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Summary

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

Abstract

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

Introduction

Mass spectrometry imaging (MSI) in microprobe mode involves desorption of the sample from a surface by a beam (laser or ions) at discrete locations over the surface of a sample. At each raster point, a mass spectrum is generated and the acquired spectra, along with the spatial location from which they were collected, can be used to simultaneously map numerous analytes within the sample. This label-free manner of imaging coupled to the sensitivity and specificity of mass spectrometry have helped MSI become one of the most rapidly evolving fields in mass spectrometry^1,2.

Matrix-assisted laser desorption/ionization (MALDI) is the mo....

Protocol

Note: The following protocol describes all the necessary steps for performing IR-MALDESI MSI experiments. In-depth details about the optimized geometry of the IR-MALDESI source and its synchronization with the laser, stage, and mass spectrometer can be found elsewhere^5,6. Animal tissue samples used in this protocol were obtained according to Institutional Animal Care and Use Committee (IACUC) and North Carolina State University regulations.

1. Tissue Preparation

1. Prepare an isopentane/dry ice bath by placing ~200 ml of isopentane in a clean beaker inside a secondary container of dry ice in a fume hood. Use protective g....

Results

The images presented in Figure 4 show the spatial distribution of metabolites in different organs in the whole-body tissue section. Unique m/z values to specific regions of the body were found using MSiReader PeakFinder, followed by batch processing for image generation. The image overlay tool (Figure 3-4) was used to align the optical image taken before ice matrix deposition with the resulting ion maps. Cholesterol is observed across all tissue .......

Discussion

The protocol above describes the key steps for performing an IR-MALDESI MSI experiment. The matrix application process (Section 3) takes approximately 20 min, which is similar to a typical matrix application process for MALDI MSI experiments by sublimation or spray-coating using a robotic sprayer. Furthermore, IR-MALDESI does not rely on partitioning of analytes into the matrix crystals⁶, and the ice matrix can be universally used for all analytes regardless of their mass, size, or chemical properties. In addi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
IR-MALDESI Source	Custom-made	N/A	Please refer to references 4 and 12 for an in-depth discussion of IR-MALDESI source development.
Q Exactive Plus	Thermo Scientific		Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer
Water, HPLC Grade	Burdick & Jackson	AH365-4
Methanol, HPLC Grade	Burdick & Jackson	AH230-4
Formic Acid	Sigma Aldrich	56302
Tunable mid-IR Laser	Opotek Inc.	IR Opolette	Tunable 2,700-3,100 nm IR OPO laser
Nitrogen Gas	Arc3 Gases	AG S-NI300-5.0	Grade 5.0 high purity nitrogen gas cylinder (300)
Cryostat	Leica Biosystems	CM 1950	Cryomicrotome
High Profile Microtome Blades	Leica Biosystems	3802123	Leica DB80HS
Mounting Medium (OCT)	Leica Biosystems	3801480	Surgipath FSC 22 mounting medium
Cryostat Specimen Disc	Leica Biosystems	14047740045	40 mm diameter
Glass Microscope Slides	VWR	48312-003	Frosted, selected, pre-cleaned