The advent of matrix-assisted laser desorption ionization has allowed the technique of mass spectrometry imaging to be used for relatively large biomolecules, enabling visualization of their location and distribution within tissues, as well as the identification of, and any changes in, disease biomarkers. Dr Shannon Cornett, applications development manager from Bruker Daltonics, discusses recent technological advances in MSI and their impact on clinical diagnostics research.

What is mass spectrometry imaging?

Mass spectrometry imaging (MSI) was originally introduced more than 50|years ago as a tool to study semiconductor surfaces. Using mass spectrometry (MS), MSI enables the visualization of the spatial distribution of molecules – biomarkers, metabolites, peptides or proteins – by their molecular masses. In practice, mass spectra are collected on an array of spatial coordinates until the entire sample is scanned. By choosing a peak in the resulting spectra that corresponds to the compound of interest, the MS data is used to map its distribution across the sample, creating pictures of the spatially resolved distribution of a compound.
In 1988, Franz Hillenkamp and Michael Karas described a method whereby they used a laser to irradiate a crystalline admixture of a biomolecule and small organic acid. Called matrix-assisted laser desorption/ionization (MALDI), they laid the foundation of a technology that opened new possibilities across science – from organic and polymer chemistry to proteomics, microbiology and drug development.
The pioneering work of Richard Caprioli and colleagues in the late 1990s demonstrated how MALDI-MS could be applied to visualize distributions of large biomolecules (such as proteins and lipids) i n cells and tissue to reveal greater insight into how molecular expression is changed by diseases like cancer. MSI can be used with different ionization techniques, including secondary ion mass spectrometry (SIMS), MALDI and desorption electrospray ionization (DESI). Today MALDI is the leading technology in clinical and biological applications of MSI, and it is widely considered the most effective for imaging tissue samples.

What are some of the current applications of MSI in clinical research?

MALDI imaging has continuously gained acceptance in clinical research. Significant technological and methodological improvements have contributed to enhance the performance of MALDI imaging recently, pushing the limits of throughput, spatial resolution and sensitivity. This has stimulated the spread of MALDI imaging across various biomedical research areas such as oncology, neurological disorders, cardiology and rheumatology, just to name a few.
MALDI imaging applications include protein characterization, glycoprotein analysis, quality control applications, polymer analysis and ultra-high throughput screening. Approximately 85% of MALDI imaging used in clinical research relates to cancer studies. Other major clinical research applications include Parkinson’s disease, Alzheimer’s, diabetes and non-alcoholic fatty liver disease, and well as detecting tumour margins.

What are the most recent technological advances in MALDI-MS imaging?

Because the information contained in each pixel is an unlabelled chemical fingerprint (or mass spectrum) of those particular cells, advances have been driven by: (a) faster acquisition of pixels; and (b) increasing the number of molecular features detected at each pixel location. Most recently, a novel combination of MALDI imaging with post-ionization (PI) has demonstrated significant enhancement in sensitivity. Studies show that this new combination, named MALDI-2, increases the sensitivity for many small molecules and lipids by up to three orders of magnitude. Further, some classes of compounds are only detectable with MALDI-2, expanding the range of applications for MALDI imaging even more. It is particularly significant for drug metabolism and pharmacokinetics (DMPK) studies.
A major limiting factor in modern drug development is use of the ‘wellstirred model’, which homogenizes organs and tissues before analysis and quantitation with liquid chromatography-mass spectrometry (LC-MS) studies. This approach is well-suited to providing exact amounts of drugs and metabolites within a target organ, but not readily compatible with pathology methods that seek to describe physiological effects of drug compounds. MALDI imaging has made a significant impact towards the conversion of plasma to tissue models by pinpointing the exact location of drugs and metabolites in tissue. The novel PI source enhances molecular imaging for pharma studies by increasing overall sensitivity, enabling quantitation for a wider range of dosing levels. Additionally, the increased variety of molecular classes expands the applicability of imaging to many more pharma projects that involve both xenobiotics and endogenous molecules.

The identification of biomarkers for disease diagnostics and prognosis is a key area of clinical research. How will these recent advances benefit biomarker identification?

Cancer cells and other diseased tissues have significant genetic and epigenetic modifications that influence the genomic expression cascade. Whether you are looking at the proteome, lipidome or metabolome, the spatial distribution of compounds contains valuable information for understanding your sample. If certain compounds are highly spatially concentrated or if molecules co-distribute in specific compartments, this vital information is lost when examining only homogenized samples. OMICS-based biomarker discovery becomes more complete when contextualized with spatial information to provide important clues into intercellular communications networks that are integral to cancer growth.
The combination of the novel MALDI-2 source mentioned previously provides the best opportunity to combine region-specific information from MALDI imaging with deep 4D OMICS coverage for biomarker discovery and molecular characterization. One advantage is the greater degree of molecular information that can be used to detect spatially significant region of interests (ROIs) in tissue sections that share common molecular signatures. For example, one study of micro-proteomic characterization of tumour subpopulations in breast cancer analysed MALDI Images of lipids to identify and target tumour subpopulations of specific molecular phenotype for laser capture microdissection (LCM). Pro¬tein extraction and tryptic digestion of small microdissected material was followed by proteomic analysis. Analysis of proteomics data of each molecular phenotype provided a more comprehensive mechanistic under¬standing of cell-type specific biological processes in situ to complement the workflow.

The resolution of near-isobaric ions has also been a challenge – what have the new technologies been able to do for imaging these types of molecules?

Quantification MSI (qMSI) remains a challenging but necessary aspect of MALDI imaging when applied to DMPK. Numerous factors such as the prevalence of isobaric endogenous compounds, chemical background from tissue matrix, and ion suppression often leads qMSI to have low reliability. However, to fully realize MALDI imaging as a versatile and highly applicable technique, quantification results must be accurate and reliable.
This is especially important in pharmacology, where the distribution of drugs and their metabolites in tissue is as important as the absolute quantification. The concentration of drugs present in disease sites determines the efficacy of the dosage and the impact of side effects, which in turn also illustrates the efficiency of any drug delivery methods. Pharmacology research is guided by determining the pharmacokinetics and the pharmacodynamics of the drug, and such studies often require screening large cohort of samples. Speed, sensitivity, spatial resolution, and specificity often determine the efficiency of a qMSI method.
The combination of the novel MALDI-2 source with timsTOF fleX can separate near-isobaric ions by their ion mobilities, significantly improving targeted compound specificity and sensitivity for quantitation in a complex molecular environment. MALDI spectra can be particularly complex in the lower mass-to-charge ratio (m/z) range owing to isobaric and near-isobaric interference from matrix ions and higher charge state analyte ions such as dimers from species to be quantified tracking of these ions that might affect the linear dynamic range. Results show that a combination of parallel accumulation and selective elution of ions by parallel accumulation– serial fragmentation (PASEF) and matching quadrupole isolation also improves sensitivity.

Mapping of steroids by traditional MSI has also been difficult. Why is it important to study steroids and how does MALDI imaging help?

Steroids are a biologically important class of compounds, and there is a growing interest in studying steroid distributions using MALDI imaging. As important components of cell membranes, steroids affect membrane fluidity and cell signalling. Hundreds of steroids can be found in plants, animals and fungi. Due to their nonpolar core structure, steroids do not ionize well by traditional MALDI imaging without specialized on-tissue derivatization protocols.
Steroids are one such analyte class that benefits strongly from this technology. We observed a sensitivity boost by up to 2–3 orders of magnitude depending on analyte and concentration [3]. It is a night-and-day difference.

What do you envisage for the future of MSI?

MALDI imaging has proven to be a powerful MS tool for mapping the distribution of molecules from a thin sample, ranging from small metabolites to large proteins, without molecular tags or labels. Application areas for MALDI imaging are diverse and growing, driven by the label-free nature of the technique and the ability to differentiate compounds by molecular weight, and also by collisional cross section.
The technology is suited for both targeted and untargeted studies. Untargeted discovery studies that use MALDI imaging are found throughout clinical research where the goal is to capitalize on the regional specificity to uncover novel biomarkers of disease and treatment. MALDI imaging is also revolutionizing pre-clinical drug discovery pipelines by providing direct distribution monitoring of targeted therapeutic compounds and their metabolites. Further, the label-free nature of the technique makes it possible to mine untargeted pharmacodynamic data from the same targeted data sets. Newer applications surrounding plants, polymers and microbes also are emerging. Eventually, we believe MALDI imaging has the potential to impact patient treatment. The technology is still evolving and we see a growing number of applications that can benefit from MALDI imaging as instrumentation continues to advance.

The interviewee

Dr Shannon Cornett PhD applications development manager
Bruker Daltonics, 40 Manning Rd, Billerica, MA 01821, USA
For further information visit Bruker Daltonics: www.bruker.com