About MALDI Imaging

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WHAT IS MALDI IMAGING?

In recent years, matrix assisted laser desorpbtion ionization (MALDI) mass spectrometry has seen many improvements applied to the technique and its applications. Whereas the focus of MALDI before has mostly been on the analysis of peptides and proteins (proteomics), the development and progress of MALDI Imaging have clearly expanded the field of application. The aim of MALDI Imaging is to provide localized information on diverse analytes in various materials. Predominantly, the examined samples are thin tissue slices of different origins.

MALDI Imaging mass spectrometry enables the visualization of the spatial distribution of proteins, peptides, pharmaceutical compounds and their metabolites, biomarkers or other compounds within thin slices of sample such as human, animal or plant tissue. It is a promising tool for putative biomarker characterization and drug development. As a first step, scientists take tissue slices mounted on microscope slides and apply a suitable MALDI matrix to the tissue, either manually or automatically.

Next, the microscope slide is inserted into a MALDI mass spectrometer. The mass spectrometer acquires mass spectra displaying the included molecular species with an instrument-dependent spatial resolution. Suitable image processing software can then be used to import data from the mass spectrometer to allow visualization and comparison with the optical image of the sample. Recent work has also demonstrated the capacity to create three-dimensional molecular images using the MALDI Imaging technology and co-registration of these image volumes to other imaging techniques such as magnetic resonance imaging (MRI).

These applications are used in biochemistry, organic chemistry, polymer chemistry, microbiology, pharmacology, clinical chemistry and forensic science.

The advantage of mass spectrometry is its ability to identify a wide diversity of molecules. Those may be small molecules such as drug candidates and their metabolites, as well as larger biomolecules such as peptides and lipids, or even intact proteins.

Due to the diversity of possible applications, MALDI Imaging now plays an important role in many scientific fields. Pharmaceutical research is a major application that can benefit from this technique.

SAMPLE PREPARATION FOR MALDI IMAGING

 CURRENTLY THE SMALLEST MALDI MATRIX CRYSTALS

– by the use of optimised spraying technique

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The sample preparation is often the limiting factor in spatial resolution. Limiting factors include crystal size and homogeneity of the matrix coverage and the undesirable migration/diffusion of analytes. In order to detect large bio-molecules such as proteins or peptides, or small molecules such as drugs or their metabolites, it is necessary to remove the lipids with a washing procedure. If the washing procedure is not performed in a fast and efficient manner, spatial resolution can be lost.In the following description we will not handle how fresh frozen or FFPE tissue samples can be cut in tiny slices or fixed on targets like standard microscope glasses, rather we will give descriptions and recommendations how matrix or enzyme solutions can be applied on tissues in order to achieve a maximum of sensitivity and resolution.

The matrix compounds are applied in water/organic solvent mixture. If a certain area of tissue remains wet longer than a few seconds during the application of matrix, the spatial resolution will be lost due to diffusion of the molecules of interest. A diffusion based migration will be higher, if:

  • the compounds of interest are small; e.g. peptides are much more affected than proteins
  • the solubility of the analytes very good in the matrix solvent. Proteins in ethanol will be less affected than smaller peptides or pharmaceuticals in aqueous organic solvents
  • the temperature is higher and the viscosity of the solvent lower

METHODS FOR MALDI IMAGING SAMPLE PREPARATION

Matrix application methods

The application method or coverage with matrix depends on the target analyte. Bio molecules such as peptides or proteins or compounds other than lipids need to be extracted. Unlike the previously mentioned compounds lipids do not need an extraction step. Lipids can be analyzed without extraction and only need an application of matrix.

TLC sprayer or other manual spray methods
Micro Spotting Technique
Ultrasonic sprayer
Modified Airbrush Technique
Electrospray Technique
Sublimation
Sublimation/Re-Crystallization

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