Sample preparation for MALDI-MS

What is MALDI Imaging?

The application of Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrometry has been changed in the recent years. Whereas before the focus of MALDI was on the analysis of peptides and proteins (proteomics), the development and progress of MALDI Imaging has clearly expanded the amount of applications. The aim of MALDI Imaging is to provide localized information on diverse analytes in various materials. The examined samples are mostly thin tissue slices of different materials. 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 samples such as human, animal or plant tissue. It is a promising tool for putative biomarker characterization and drug development.
For gaining usable results in the process of MALDI Imaging the sample preparation is crucial. Essential is how the matrix is applied onto the sample.


Crystal size of the matrix in MALDI Imaging

 Currently the smallest MALDI matrix crystals worldwide !


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

Influence of crystal size on resolution in MALDI Imaging

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.


Why a graduated spray procedure is so important

 Other coverage methods

TLC sprayer or other manual spray methods

This simple and cheap method requires some experience and skill for a minimum homogeneity of the matrix layers. Even then the drop size is not uniform; as well as very small drops, the sprayed matrix layer will contain also large droplets; see picture below. This method is not suitable for a single cell analysis, which requires crystal size about 1 micron. The spray results of so called airbrush sprayer are much better; but application of a small number of layers requires a highly skilled operator to achieve acceptable homogeneity. A number of authors have reported copper adducts resulting from the brass metal of the paintbrush sprayer. The standard microscope slide matrix coated with a TLC sprayer shows how unfavorable inhomogeneous with very unequal drop sizes the result is.

Micro Spotting Technique

This places pico liter size drops on the tissue surface. Frequently it was reported that the spray nozzle clogged, especially when concentrated matrix solutions were used. Spot diameters are up to 150 µm and spot distances between 20 and 250 µm are typical. These figures show that the technique is not suitable for a single cell analysis and the highest spatial resolution is not lower than 200 µm. Whether this small fluid volume would be sufficient for a satisfactory extraction of macro molecules must be evaluated. An additional problem may arise when the second layer drops do not hit exactly the same position as of the last layer. This would worsen the spatial resolution furthermore. This technique is used by Portrait® (Labcyte Inc., USA) and ChIP-1000® (Shimadzu Corp. Japan)

Ultrasonic sprayer

This generates fine droplets of different sizes from a matrix solution which is sprayed into a chamber. Each layer is dried with a flow of nitrogen. A light scattering detector in the middle of the chamber generates information about the dryness and thickness of the matrix layer. The disadvantage of this method is that different drops sizes are deposited in different regions. So bigger drops will be placed near to the ultrasonic source and smaller drops on the opposite side of the chamber. This results in the droplet size and matrix concentration within the chamber being inhomogeneous. The longer waiting times between the layers and the time to achieve the total coverage ebbing in the range of hours leads to the crystal size being >20 µm for CHCA and even larger for DHB. An example of this technique would be the ImagePrep® (Bruker Daltronics; Germany)

Modified Airbrush Technique

This technique uses an optimized spray generator to produce small matrix droplet sizes. There are currently 2 vendors of these types of instruments. These instruments ‘atomize’ the matrix solution to produce very small drops with a compressed air or nitrogen flow. One instrument heats the solution just before spraying in order to increase the matrix concentration. This can be of advantage for CHCA solutions since it has limited solubility in ACN/water mixtures – typically a maximum concentration of 16 mg/mL. Both instruments move the spray head over a surface area which has been defined by the user. Small droplet size and uniform movement result in a homogeneous coating. One of the differences between these two instruments is the method of matrix delivery to the spray head. In one instrument a modern syringe pump delivers solutions, precisely at low flow rates down to the pico liter range. The low flow rate means that the usage of expensive enzyme solution is kept to a minimum. The other instrument uses a small and simple HPLC pump with high dead volume; this requires a large volume of expensive enzyme solution. The extraction efficiency of both instruments is excellent especially for bio molecules; the total number of layers needed for a sufficient coverage and the speed of application are also very good. Typically the time to apply several layers would be minutes rather than hours. The HTX-Sprayer®(HTX Technologies, LLC, USA) and SunCollect® (SunChrom, Germany) are instruments using this technique.

Electrospray Technique

Instruments using this technique spray the matrix solution with a spray needle, where an electrical field is built-up between spray needle and target. It can be used for all compounds such as Proteins / Peptides and small molecules. A constant potential field is essential for a homogeneous coating. A number of authors are reporting an extended period of time for the matrix to become dried with no user control of this process.


creates significantly smaller crystals and homogeneous coating on tissues with a number of matrix compounds, but not all of them. Since the matrix crystallizes directly from the gas phase on the tissue, the matrix will be also purified by this procedure. One disadvantage is that there will be no extraction, so this method cannot be used for Peptides/Proteins or small molecules. In this method the matrix does not desorb into the tissue but instead sits as an independent layer on top of the tissue. Therefore this method can only be used for lipids or few small molecules, which are highly evaporative. The instrumental design is relatively simple and also the deposition time is very short – approx. 15 minutes. The matrix coverage is applied to the whole target and cannot be directed onto a special area. The matrix consumption is relatively high and can be expensive. There are only a few suitable matrices for this technique e.g. DHB (150 °C) and CHCA (170 °C) and also DAN. This method cannot be used for other matrices or enzymes.


tries to eliminate the lack of extraction by keeping the sublimated layer under saturated humidity for a very long period in order to achieve small extraction efficiency. The target or microscope slide with a tissue section is typically kept for 24-72 hours in a high humidity environment. Usually the extraction efficiency will be not optimal and the time requirement is extremely long.

SunCollect System for MALDI Imaging

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