Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful analytical technique that enjoys the sensitivity of mass spectrometry coupled with spatial distribution information.1 Since its inception only a couple of decades ago,2 it has made tremendous technological leaps and discoveries in the biological sciences.3 A raster of the MALDI mass spectrometry works by applying a matrix to a sample, which aids in the ionization of sample molecules upon excitation with a laser. The mixture of ionized molecules is funneled into the mass spectrometer, where their masses are measured and recorded.
In the application of MALDI IMS, the sample can be a tissue specimen, for example, and matrix can either be robotically sprayed over the tissue or individual matrix spots can be deposited across the tissue surface in a two-dimensional (2D) grid.2 A raster of the laser beam is performed over the tissue specimen, sampling multiplex spots’ or pixels and rendering a morphological 2D image of ion profiles, i.e., all detectable ions within a matrix spot, across the tissue specimen.2 “The bioanalysis of tissue samples is important to advance research, but it was apparent to us that not all biological processes occurred in the same place and time. The spatiotemporal aspect was missing, and we wanted to capture that. It occurred to us that the mass spectrometer laser could be modified to raster over a tissue sample,” recalls Professor Richard Caprioli, the Stanford Moore Chair at the Department of Biochemistry, Director of the Mass spectrometry Research Center at Vanderbilt University, and author of that seminal report. “There were plenty of steps along the way because the instrument wasn’t designed to do that, but out of our modifications and improvements an effective molecular microscope emerged.”
MALDI IMS boasts several advantages over conventional methods of tissue analysis. It is untargeted, and consequently doesn’t require a priori selection of a protein analyte, as, for example, by antibody detection of a specific protein by immunohistochemistry (IHC). Moreover, it is completely multiplexed because it detects all ions from a tissue sample in contrast to IHC, which is limited by the number of fluorophore colors and antibody combinations that can be simultaneously used or contiguous tissue sections that can be serially compared for certain proteins. MALDI IMS is an in situ, label-free technique, and since it performs a molecular analysis, it provides information on the cumulative effect of genomic/epigenomic changes, which has important consequences in disease pathology in heterogenous biological systems. Finally, the MALDI laser typically does not ablate all of the tissue and the specimen can be reexamined.4
MALDI IMS: sample applications
The number of applications MALDI IMS has been applied to continue to emerge. Analysis of tumor tissue pathology ranks high on the list,3-5 for both diagnostic, i.e., differentiating tumor tissue from normal healthy tissue, and prognostic applications, i.e., identifying tumor tissue from patients that will respond to therapy or their prognosis. The list also includes neuroproteomics and neurodegenerative disease,6 and tissue composition, such as histone modifications7 and phospholipid8 distribution, in addition to analysis of signaling pathways.9 MALDI IMS can also advance drug pharmacokinetics and development, by studying the fate of drugs, both in their tissue distribution and metabolic breakdown.10 MALDI IMS has also made inroads in plant biology11 and environmental toxicology,12 assessing risk and adverse effects from pollutants and their possible mechanism of action. Recently, the image analysis of low molecular weight metabolites has been reported.13,14
A recent and exciting application of MALDI IMS uncovered biomarkers of the Pseudomonas aeruginosa infection in situ in burn wounds from mice.15 “We hypothesized that bacteria would have a different chemical ‘signature’ than skin cells so MALDI IMS was a logical way to test our idea,” elaborated Professor Brian Bothner, at the Department of Chemistry and Biochemistry, Montana State University of his work. “Being able to detect the presence, location, and type of bacteria in a wound has significant clinical application.” The method, if widely applicable, could shave hours to days compared to MALDI-TOF for microbial identification, which presently requires a lengthy culturing step. In contrast, “the imaging approach (MALDI IMS) was made directly on the tissue biopsy and the analysis was accomplished within minutes,” Professor Bothner explained.
MALDI IMS: methodologies and modalities
Several considerations are necessary to prepare MALDI IMS samples, including section thickness, tissue washes, and matrix selection and application, which are generally optimized empirically for each new experiment.1 “It was fairly straight-forward to initiate experiments in our laboratory and to collect the data,” Professor Bothner said of the technology. “The most technical aspect was applying the matrix to the sample, which eventually succeeded with a standard airbrush used for painting. A number of matrix products are now commercially available and many MALDI-TOF instruments now come with imaging software already integrated as a ready-to-use application.”
The scope of analytes detected by MALDI IMS is broad, encompassing proteins (including post-translationally modified, cleaved, or degraded proteins) in addition to peptides, lipids and phospholipids, and small molecules, such as metabolites and drugs.3,4 MALDI IMS is flexible on the type of specimen, and can range from tissue sections, to whole organ or organism sections of smaller animals, to tissue microarrays.3,10 Fresh frozen, formalin-fixed and paraffin-embedded (FFPE) tissue with antigen retrieval can all be used as specimens.3 It is also possible to reconstruct serial sections to render 3D images.6 The requirements of the experiment dictate the resolution needed. High-resolution necessitates matrix sublimation, which compromises on the sensitivity, requires longer collection time and leads to large data sets.1
For its diagnostic and prognostic applications,3 it is not necessary to identify proteins. Rather, multivariate analysis can be applied to the mass ion profiles of experimental (e.g., tumor) versus control (e.g., healthy) tissue to extract their defining mass spectrum features.1,3 Prediction models can then be built to differentiate tumor from healthy tissue to diagnose cancer or to predict patient prognosis based on distinctive mass ion biomarkers. “The detected proteins don’t necessarily need to be related to the disease,” explains Professor Caprioli. “They just need to be specific and unique to the disease to identify a ‘disease state’.” Protein identification is also possible, but challenging, and includes comparison to MALDI on tissue homogenate or by in situ, on-section, tryptic digest;1 however, novel microextraction methods are also being developed.16 Quantification of data is also achievable if care is taken to account for several caveats,1,4,10 and collected data can be represented in spectral format and as color-coded images.1
MALDI IMS: bench-to-bedside on the horizon
MALDI-TOF for microbial identification has made the transition from research to clinical applications, what of MALDI IMS? “The technique is well validated,” Professor Caprioli says of the technique he brought to life. “Several issues still need to be addressed, such as bringing pathologists and healthcare professionals on board, but the technique stands to significantly improve healthcare for patients.” Ultimately, a translation to the clinic will require automated protocols and high-throughput capabilities, in addition to quantification. “Standardization and certification protocols are in progress,” adds Professor Caprioli, “Careful methodology will be necessary to minimize variation and deliver high quality results to aid in diagnosis.” Professor Bothner also foresees potential clinical applications, “This technology could be placed directly in a clinic so that doctors could determine the extent of microbial infection in tissue and hopefully even identify the type of bacteria.”
(1) Gessel, M. M.; Norris, J. L.; Caprioli, R. M. MALDI imaging mass spectrometry: spatial molecular analysis to enable a new age of discovery. J Proteomics 2014, 107, 71.
(2) Caprioli, R. M.; Farmer, T. B.; Gile, J. Molecular imaging of biological samples: localization of peptides and proteins using MALDI-TOF MS. Anal Chem 1997, 69 (23), 4751.
(3) Schwamborn, K.; Caprioli, R. M. MALDI imaging mass spectrometry--painting molecular pictures. Mol Oncol 2010, 4 (6), 529.
(4) Aichler, M.; Walch, A. MALDI Imaging mass spectrometry: current frontiers and perspectives in pathology research and practice. Lab Invest 2015, 95 (4), 422.
(5) Kriegsmann, J.; Casadonte, R.; Kriegsmann, K.; Longuespee, R.; Kriegsmann, M. Mass spectrometry in pathology - Vision for a future workflow. Pathol Res Pract 2018.
(6) Andersson, M.; Andren, P.; Caprioli, R. M. In Neuroproteomics; Alzate, O., Ed.; CRC Press/Taylor & Francis Llc.: Boca Raton (FL), 2010.
(7) Lahiri, S.; Sun, N.; Buck, A.; Imhof, A.; Walch, A. MALDI imaging mass spectrometry as a novel tool for detecting histone modifications in clinical tissue samples. Expert Rev Proteomics 2016, 13 (3), 275.
(8) Sparvero, L. J.; Amoscato, A. A.; Dixon, C. E.; Long, J. B.; Kochanek, P. M.; Pitt, B. R.; Bayir, H.; Kagan, V. E. Mapping of phospholipids by MALDI imaging (MALDI-MSI): realities and expectations. Chem Phys Lipids 2012, 165 (5), 545.
(9) Franck, J.; Arafah, K.; Elayed, M.; Bonnel, D.; Vergara, D.; Jacquet, A.; Vinatier, D.; Wisztorski, M.; Day, R.; Fournier, I.et al. MALDI imaging mass spectrometry: state of the art technology in clinical proteomics. Mol Cell Proteomics 2009, 8 (9), 2023.
(10) Castellino, S.; Groseclose, M. R.; Wagner, D. MALDI imaging mass spectrometry: bridging biology and chemistry in drug development. Bioanalysis 2011, 3 (21), 2427.
(11) Kaspar, S.; Peukert, M.; Svatos, A.; Matros, A.; Mock, H. P. MALDI-imaging mass spectrometry - An emerging technique in plant biology. Proteomics 2011, 11 (9), 1840.
(12) Lagarrigue, M.; Caprioli, R. M.; Pineau, C. Potential of MALDI imaging for the toxicological evaluation of environmental pollutants. J Proteomics 2016, 144, 133.
(13) Kleinridders, A.; Ferris, H. A.; Reyzer, M. L.; Rath, M.; Soto, M.; Manier, M. L.; Spraggins, J.; Yang, Z.; Stanton, R. C.; Caprioli, R. M.et al. Regional differences in brain glucose metabolism determined by imaging mass spectrometry. Mol Metab 2018, 12, 113.
(14) Soto, M.; Orliaguet, L.; Reyzer, M. L.; Manier, M. L.; Caprioli, R. M.; Kahn, C. R. Pyruvate induces torpor in obese mice. Proc Natl Acad Sci U S A 2018, 115 (4), 810.
(15) Hamerly, T.; Everett, J. A.; Paris, N.; Fisher, S. T.; Karunamurthy, A.; James, G. A.; Rumbaugh, K. P.; Rhoads, D. D.; Bothner, B. Detection of Pseudomonas aeruginosa biomarkers from thermally injured mice in situ using imaging mass spectrometry. Anal Biochem 2017, 539, 144.
(16) Ryan, D. J.; Nei, D.; Prentice, B. M.; Rose, K. L.; Caprioli, R. M.; Spraggins, J. M. Protein identification in imaging mass spectrometry through spatially targeted liquid micro-extractions. Rapid Commun Mass Spectrom 2018, 32 (5), 442.