This is the first post in our technology focused series, “The Bridge to Improved Patient Outcomes.” In this post, I would like to introduce you to matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging technology, a field in which I have been working for over 14 years. The purpose of this post is to introduce you to the basics of mass spectrometry imaging (MSI), which will serve as a building block for future posts in the series.
Let’s start with a step-wise discussion about how MSI is performed:
Step 1: Tissue Specimen
MSI uses thin tissue sections, similar to those a pathologist uses to evaluate a biopsy, except that the MSI tissue section is collected onto a specialized target than can be used in a MALDI mass spectrometer. MSI can be performed on both fresh frozen and formalin fixed, paraffin embedded (FFPE) tissue specimens. However, FFPE tissue has almost exclusively been used for the detection of tryptic peptides as proteins become crosslinked during the fixation, preventing them from being detected intact and most small molecules such as lipids and metabolites are lost either during fixation or deparaffinization methods. MALDI profiling can also be carried out on cytologic specimens and biofluids that are directly related to a specific disease, but that will be a topic for another post.
Step 2: Specimen Preparation
Appropriate specimen preparation is carried out for the target molecular class (protein, peptide, lipid, or drug and metabolite) of interest, as discussed below. During this process, a matrix, typically a small organic acid, is applied to the surface of the specimen, usually in a solvent that helps to extract and co-crystallize the analytes of interest with the matrix. The matrix can be applied using robotic sprayers, sublimation, or manually using an airbrush or thin layer chromatography sprayer.
Step 3: MALDI Mass Spectrometer
The specimen is interrogated in the mass spectrometer using a UV laser that is fired at the tissue in an ordered array of regular spacing. The matrix absorbs the laser light and acts to desorb and ionize the molecules from the tissue surface. In time-of-flight analysis, ionized molecules are accelerated with high voltage and travel through a flight tube under high vacuum until the ionized molecules arrive at a detector at the end of the flight tube. Since all ions are accelerated with the same energy, those that are smaller will fly faster, while those that are larger will fly slower, across the fixed distance. By measuring the time it takes for the ions to reach the detector, and through the use of calibration standards of known molecular weight, the mass-to-charge ratio of the ions can be determined.
A full spectrum is collected at each targeted location in the tissue. This can be likened to the pixels in a picture or on your television, except that instead of having three color channels (red, green, and blue), hundreds to thousands of biomolecules are detected. Any one of these biomolecules can be displayed as a false color image indicating its spatial localization and relative intensity across the surface of the tissue section. More sophisticated statistical analysis can be carried out on this spectral data, and we will address this topic later in the series.
Now that we have discussed the MSI process, let's detail the types of projects where this technology is most applicable:
Project Type 1: Intact Protein Analysis
Intact protein analysis is frequently carried out on preclinical specimens and animal tissues where it is not customary to perform formalin fixation which results in crosslinking of proteins, thus necessitating digestion. Intact protein analysis can be beneficial when trying to map localization of posttranslational modifications on proteins (e.g., methylation and acetylation on histones) that can be altered with disease state. This type of analysis is also commonly used for cytology and biofluids.
Intact protein analysis must be carried out on fresh frozen tissue specimens. Sections are typically 12 µm thick and are usually fixed with graded alcohol to remove biological salts and lipids that can suppress protein signal. Sinapinic acid or super dihydroxybenzoic acid (super DHB) are the matrices of choice for protein analysis. The matrices are typically applied in 50-90% acetonitrile with 0.1-1% trifluoroacetic acid (TFA). For drier applications (i.e., high organic content or sublimation), a recrystallization step is often performed by sealing the matrix coated slide in a petri dish with a humidified environment of organic solvent at elevated temperature for a few minutes. This helps to extract the proteins into the crystal layer so that they can be detected in the mass spectrometer. Proteins analyzed are typically small, with molecular weights ranging from 2 kDa to 30 kDa and detected in positive ion mode.
Project Type 2: Peptide Analysis
Peptide analysis involves more sample preparation than the other types of analysis and is the method of choice for most clinical specimens that are banked as FFPE tissue blocks. The fixation process crosslinks proteins, requiring that they be digested so that peptides that are not crosslinked can be analyzed. Specimens used may be traditional biopsies or surgical specimens as well as tissue microarrays for high throughput analysis.
Peptide analysis, through use of on-tissue tryptic digestion, has mostly been demonstrated in FFPE tissue, although examples in fresh frozen tissue have been reported. Sections are typically 5 µm thick and are subjected to deparaffinization and rehydration with xylene and graded alcohol followed by heat induced antigen retrieval. Subsequently, trypsin is applied via a robotic sprayer to allow for in situ enzymatic digestion of the proteins, a necessary step due to protein crosslinking that occurs during the formalin fixation process. The sections are incubated in a humidified environment for several hours to maximize digestion efficiency while minimizing delocalization. Finally, α-cyano-4-hydroxycinnamic acid (CHCA) matrix is applied to the section in 50-70% acetonitrile with 0.1% TFA. No recrystallization is needed, and peptides are generally detected in positive ion mode.
Project Type 3: Lipid Analysis
Lipid analysis is used in a wide range of applications from preclinical and animal models through clinical and intra-operative specimens. Lipids are generally the easiest biomolecules to analyze, and many can be detected without the use of solvent in the matrix application.
Lipid analysis must be carried out on fresh frozen tissue specimens. Sections are typically 12 µm thick, and the sections may be washed with cold ammonium formate to remove sodium and potassium ions from the sections that can cause adducts to form on the lipid species, complicating the analysis. DHB or dihydroxyacetophenone (DHA) are matrices typically used for positive and negative ion mode analysis, respectively. Matrix may be applied in 50% methanol or via dry application such as sublimation with no need for recrystallization. Intact lipids, along with lysolipids, are readily detected, with different lipid classes being detected in positive and negative ion mode.
Project Type 4: Drug & Metabolite Analysis
Drug and metabolite analyses have most frequently been used to determine if a drug and/or its downstream metabolites are making it to target locations in an animal. This is typically done in preclinical, animal models in pharmaceutical or research settings. An animal is dosed with the compound of interest and sacrificed after a specified time period. The drug and metabolites are then mapped throughout the target tissue or whole animal in either a qualitative or quantitative manner. This approach is much more molecule specific than traditional quantitative whole-body autoradiography approaches that cannot distinguish between drug and metabolite.
Drug and metabolite analysis have been demonstrated in both fresh frozen and FFPE tissue, although only polar metabolites have been detected in FFPE tissue. Sections are typically 12 µm thick for frozen tissue and 5 µm thick for FFPE with washing avoided to prevent delocalization or loss of analytes. FFPE sections are subjected only to a xylene rinse to remove the paraffin wax from the sample. Matrix and solvent choices are highly variable depending on the target molecules of interest, often requiring extensive method optimization or the use of MS/MS to confirm identity. Drugs and metabolites may be detected in either positive or negative ion mode.
If you would like to learn more about MSI, please refer to the following review articles. If you have additional questions about MSI, I invite you to comment at the bottom of this post.
- Imaging of intact tissue sections: moving beyond the microscope
- MALDI imaging mass spectrometry of human tissue: method challenges and clinical perspectives
- MALDI imaging mass spectrometry: bridging biology and chemistry in drug development
- Review of matrix-assisted laser desorption ionization-imaging mass spectrometry for lipid biochemical histopathology
Our next technology focused post will explore the process of Histology Guided Mass Spectrometry (HGMS) Profiling for Clinical Applications. HGMS is a specialized application of MSI that is ideally suited to clinical application research projects.