Credit: The Royal Society of Chemistry

One of the most widely utilized techniques in mass spectrometry imaging (MSI) is label-free MALDI (Matrix-Assisted Laser Desorption Ionization), which can be coupled to a variety of mass analyzers to create brilliant molecular maps. Application targets include lipids, proteins, peptides, drugs, and their metabolites, says Gregory Roman, PhD, Sr. Scientist, Waters Corporation.

MALDI can commonly be used to analyze a wide range of molecular weights, ranging from small molecules to peptides and proteins. Large proteins and antibodies with masses greater than 50 kDa have been observed (1).

The technique necessitates the addition of a matrix that assists in the ionization of a given set of analytes. Some common matrix components include alpha-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), and 2,5-dihydroxybenzoic acid (DHB).  A plethora of alternative matrix techniques exist with novel methods developed annually.

The necessity of a MALDI matrix application can be a source of sample variation. Analyte diffusion through the thin MALDI matrix layer can slightly reduce spatial resolution. It can be argued, Roman inserts, that one of the most important steps in the MALDI MSI workflow is the matrix deposition and the associated sample preparation.

Main MALDI Matrix Selection Considerations 

According to Roman, a number of practical parameters are pertinent when considering a matrix for MALDI. These include sublimation rate within the MSI instrument, thickness and consistency of the matrix layer, spectral overlap between the matrix and analyte of interest, and available application approach.

Appropriate matrixes should be matched with the target analyte, for example: proteins – SA, peptides –CHCA, lipids – DHB, CHCA, DHAP, THAP, p-nitroanaline, and small molecules – DHB, CHCA, SA (2).

When choosing an application approach, Roman says, options include manual sprayers, such as an air brush, or sprayer instruments with established protocols. These methods can either deposit a spot or a homogeneous coating on the surface of the tissue section for imaging experiments.

Sample Stabilization and Preparation

When considering the workflow for a MALDI matrix application it is important to consider the effects of both analyte degradation and delocalization. Roman highly recommends stabilizing the sample to reduce the variation of analyte abundance following postmortem collection. Stabilization methods can include flash freezing, formalin fixation, microwave irradiation or heat stabilization (3).

Sample preparation can be challenging and necessitates some trial and error, cautions Roman.  For example, rapidly degrading neurotransmitters have been stabilized with in situ freezing, while heat stabilization has been demonstrated to show mild improvement for proteins, peptides, and some lipids.

In addition, enzymatic activity can degrade proteins, resulting in fewer proteins and more peptides. Changes in post-translational modification can also manifest in degradation patterns. Both flash freezing and formalin fixation can remedy these challenges.

Cryosectioning is part art, part science rues Roman. The quality of the sectioning can greatly determine the quality of the retrieved images and can be a heavy manual burden in trial and error to get “good sections”. For instance, dialing in the correct temperature, angle of the sectioning block, and the quality of the blade, can all be factors that influence the quality. For the novice, this requires a lot of patience.

After cryosectioning, in many cases, some sample washing prior to applying the matrix can be useful to remove interferences, such as salts and lipids, reduce signal suppression and adduct formation, improve on-tissue crystallization and, possibly, provide better sensitivity. The trade off, adds Roman, is that washing may induce delocalization of the analyte and loss of soluble analytes on the surface.

Critical Experimental Parameters

When developing a procedure from sample collection through analysis one should be cognizant of minimizing sample degradation, physical deformation, and analyte delocalization, while optimizing sensitivity or resolution. Keeping these goals in mind when developing a MALDI workflow is critical to success, emphasizes Roman. Many parameters can be difficult to control and there will be tradeoffs.

Novices should keep in mind that there is no single MALDI solution for all analytes. It takes time to develop a method that is appropriate for the tissue type and analyte of interest, says Roman. When planning an imaging experiment, ensure that you have identified your sample, the factors that affect it, and the overall goal of the experiment.

At the outset, MALDI can seem like a daunting task of sample prep and matrix application that is filled with a mine field of hazards. Rest assured, says Roman, that given some patience, an eye to detail, well defined goals and target analytes, it is possible to build a method capable of generating brilliant molecular maps.



  1. Chaurand and Caprioli, Electrophoresis. 23. 3125 (2002)
  2. Caprioli, et al., Advanced Imaging Mass Spectrometry (AIMS) Short Course and Workshop (2018), Nashville TN
  3. Cecilia, E., Noritaka, M., Ikuko, Y., Takahiro, H., Mitsutoshi, S., MALDI Imaging Mass Spectrometry- A Mini Review of Methods and Recent Developments, MS Society of Japan, (2013).
Previous articleVaccine Study Explores COVID-19 and Autoimmune Disease
Next articleResearchers Discover the Power behind Innate Lymphoid Cells
Previous articleVaccine Study Explores COVID-19 and Autoimmune Disease
Next articleResearchers Discover the Power behind Innate Lymphoid Cells