Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is emerging as a powerful tool for investigating the distribution of molecules within biological systems through the direct analysis of thin tissue sections. Unique among imaging methods, MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement. We discuss the current state of the art of MALDI-IMS along with some recent applications and technological developments that illustrate not only its current capabilities but also the future potential of the technique to provide a better understanding of the underlying molecular mechanisms of biological processes.
Matrix sublimation has demonstrated to be a powerful approach for high-resolution matrix-assisted laser desorption ionization (MALDI) imaging of lipids, providing very homogeneous solvent-free deposition. This work presents a comprehensive study aiming to evaluate current and novel matrix candidates for high spatial resolution MALDI imaging mass spectrometry of lipids from tissue section after deposition by sublimation. For this purpose, 12 matrices including 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA), 2,6-dihydroxyacetphenone (DHA), 2',4',6'-trihydroxyacetophenone (THAP), 3-hydroxypicolinic acid (3-HPA), 1,8-bis(dimethylamino)naphthalene (DMAN), 1,8,9-anthracentriol (DIT), 1,5-diaminonapthalene (DAN), p-nitroaniline (NIT), 9-aminoacridine (9-AA), and 2-mercaptobenzothiazole (MBT) were investigated for lipid detection efficiency in both positive and negative ionization modes, matrix interferences, and stability under vacuum. For the most relevant matrices, ion maps of the different lipid species were obtained from tissue sections at high spatial resolution and the detected peaks were characterized by matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. First proposed for imaging mass spectrometry (IMS) after sublimation, DAN has demonstrated to be of high efficiency providing rich lipid signatures in both positive and negative polarities with high vacuum stability and sub-20 μm resolution capacity. Ion images from adult mouse brain were generated with a 10 μm scanning resolution. Furthermore, ion images from adult mouse brain and whole-body fish tissue sections were also acquired in both polarity modes from the same tissue section at 100 μm spatial resolution. Sublimation of DAN represents an interesting approach to improve information with respect to currently employed matrices providing a deeper analysis of the lipidome by IMS.
MALDI (matrix-assisted laser desorption/ionization) imaging mass spectrometry (IMS) is a new technology that generates molecular profiles and two-dimensional ion density maps of peptide and protein signals directly from the surface of thin tissue sections. This allows specific information to be obtained on the relative abundance and spatial distribution of proteins. One important aspect of this is the opportunity to correlate these specific ion images with histological features observed by optical microscopy. To facilitate this, we have developed protocols that allow MALDI mass spectrometry imaging and optical microscopy to be performed on the same section. Key components of these protocols involve the use of conductive glass slides as sample support for the tissue sections and MS-friendly tissue staining protocols. We show the effectiveness of these with protein standards and with several types of tissue sections. Although stain-specific intensity variations occur, the overall protein pattern and spectrum quality remain consistent between stained and control tissue samples. Furthermore, imaging mass spectrometry experiments performed on stained sections showed good image quality with minimal delocalization of proteins resulting from the staining protocols.
A novel method for high-throughput proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue microarrays (TMA) is described using on-tissue tryptic digestion followed by MALDI imaging MS. A TMA section containing 112 needle core biopsies from lung-tumor patients was analyzed using MS and the data were correlated to a serial hematoxylin and eosin (H&E)-stained section having various histological regions marked, including cancer, non-cancer, and normal ones. By correlating each mass spectrum to a defined histological region, statistical classification models were generated that can sufficiently distinguish biopsies from adenocarcinoma from squamous cell carcinoma biopsies. These classification models were built using a training set of biopsies in the TMA and were then validated on the remaining biopsies. Peptide markers of interest were identified directly from the TMA section using MALDI MS/MS sequence analysis. The ability to detect and characterize tumor marker proteins for a large cohort of FFPE samples in a high-throughput approach will be of significant benefit not only to investigators studying tumor biology, but also to clinicians for diagnostic and prognostic purposes.
Organic secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry can be used to produce molecular images of samples. This is achieved through ionization from a clearly identified point on a flat sample, and performing a raster of the sample by moving the point of ionization over the sample surface. The unique analytical capabilities of mass spectrometry for mapping a variety of biological samples at the tissue level are discussed. SIMS provides information on the spatial distribution of the elements and low molecular mass compounds as well as molecular structures on these compounds, while MALDI yields spatial information about higher molecular mass compounds, including their distributions in tissues at very low levels, as well as information on the molecular structures of these compounds. Application of these methods to analytical problems requires appropriate instrumentation, sample preparation methodology, and a data presentation usually in a three-coordinate plot where x and y are physical dimensions of the sample and z is the signal amplitude. The use of imaging mass spectrometry is illustrated with several biological systems.
MALDI imaging mass spectrometry (IMS) has become a valuable tool for the investigation of the content and distribution of molecular species in tissue specimens. Numerous methodological improvements have been made to optimize tissue section preparation and matrix deposition protocols, as well as MS data acquisition and processing. In particular for proteomic analyses, washing the tissue sections before matrix deposition has proven useful to improve spectral qualities by increasing ion yields and the number of signals observed. We systematically explore here the effects of several solvent combinations for washing tissue sections. To minimize experimental variability, all of the measurements were performed on serial sections cut from a single mouse liver tissue block. Several other key steps of the process such as matrix deposition and MS data acquisition and processing have also been automated or standardized. To assess efficacy, after each washing procedure the total ion current and number of peaks were counted from the resulting protein profiles. These results were correlated to on-tissue measurements obtained for lipids. Using similar approaches, several selected washing procedures were also tested for their ability to extend the lifetime as well as revive previously cut tissue sections. The effects of these washes on automated matrix deposition and crystallization behavior as well as their ability to preserve tissue histology were also studied. Finally, in a full-scale IMS study, these washing procedures were tested on a human renal cell carcinoma
Molecular imaging of tissue by MALDI mass spectrometry is a powerful tool for visualizing the spatial distribution of constituent analytes with high molecular specificity. Although the technique is relatively young, it has already contributed to the understanding of many diverse areas of human health. In recent years, a great many advances in the practice of imaging mass spectrometry have taken place, making the technique more sensitive, robust, and ultimately useful. The purpose of this review is to highlight some of the more recent technological advances that have improved the efficiency of imaging mass spectrometry for clinical applications. Advances in the way MALDI mass spectrometry is integrated with histology, improved methods for automation, and better tools for data analysis are outlined in this review. Refined top-down strategies for the identification and validation of candidate biomarkers found in tissue sections are discussed. A clinical example highlighting the application of these methods to a cohort of clinical samples is described.
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