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Alzheimer's disease (AD) is the most common form of dementia; however, at the present time there is no disease-modifying drug for AD. There is increasing evidence supporting the role of lipid changes in the process of normal cognitive aging and in the etiology of age-related neurodegenerative diseases. AD is characterized by the presence of intraneuronal protein clusters and extracellular aggregates of β-amyloid (Aβ). Disrupted Aβ kinetics may activate intracellular signaling pathways, including tau hyperphosphorylation and proinflammatory pathways. We analyzed and visualized the lipid profiles of mouse brains using MALDI-TOF MS. Direct tissue analysis by MALDI-TOF imaging MS (IMS) can determine the relative abundance and spatial distribution of specific lipids in different tissues. We used 5XFAD mice that almost exclusively generate and rapidly accumulate massive cerebral levels of Aβ-42 (1). Our data showed changes in lipid distribution in the mouse frontal cortex, hippocampus, and subiculum, where Aβ plaques are first generated in AD. Our results suggest that MALDI-IMS is a powerful tool for analyzing the distribution of various phospholipids and that this application might provide novel insight into the prediction of disease.

Neuronal membrane phospholipids are highly affected by oxidative stress caused by ischemic injury. Thus, it is necessary to identify key lipid components that show changes during ischemia to develop an effective approach to prevent brain damage from ischemic injury. The recent development of MALDI imaging MS (MALDI IMS) makes it possible to identify phospholipids that change between damaged and normal regions directly from tissues. In this study, we conducted IMS on rat brains damaged by ischemic injury and detected various phospholipids that showed unique distributions between normal and damaged areas of the brain. Among them, we confirmed changes in phospholipids such as lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin by MALDI IMS followed by MS/MS analysis. These lipids were present in high concentrations in the brain and are important for maintenance of cellular structure as well as production of second messengers for cellular signal transduction. Our results emphasize the identification of phospholipid markers for ischemic injury and successfully identified several distinctly located phospholipids in ischemic brain tissue.

Since the emergence of proteomics methods, many proteins specific for renal cell carcinoma (RCC) have been identified. Despite their usefulness for the specific diagnosis of RCC, such proteins do not provide spatial information on the diseased tissue. Therefore, the identification of cancer-specific proteins that include information on their specific location is needed. Recently, matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) based imaging mass spectrometry (IMS) has emerged as a new tool for the analysis of spatial distribution as well as identification of either proteins or small molecules in tissues. In this report, surgical tissue sections of papillary RCC were analyzed using MALDI-IMS. Statistical analysis revealed several discriminative cancer-specific m/z-species between normal and diseased tissues. Among these m/z-species, two particular proteins, S100A11 and ferritin light chain, which are specific for papillary RCC cancer regions, were successfully identified using LC-MS/MS following protein extraction from independent RCC samples. The expressions of S100A11 and ferritin light chain were further validated by immunohistochemistry of human tissues and tissue microarrays (TMAs) of RCC. In conclusion, MALDI-IMS followed by LC-MS/MS analysis in human tissue identified that S100A11 and ferritin light chain are differentially expressed proteins in papillary RCC cancer regions.

Phospholipids (PLs) are the major building block molecules of cellular membranes. Their composition varies depending on cell types and cellular compartments. Thus, the information regarding PL distribution in tissue has important physiological and pathological significance. Recent developments in imaging mass spectrometry (IMS) have allowed complete mapping of the PL species on tissue. The IMS technique can detect different classes of PLs as well as their location information directly from tissue sections. PL head groups carry either positive and/or negative charges; therefore, IMS experiments must be conducted in both positive- and negative-ion mode to detect all types of phospholipids. Several conventional matrixes were applied on tissue for better identification. This study was conducted to enable appropriate matrix selection and optimized matrix preparation for IMS experiments in both ion modes that maximize PL identification from a single brain tissue section. The optimized matrix 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA) with a mixture of trifluoroacetic acid (TFA) and piperidine as ion pairing agents showed improved stability and consistency during both ion mode experiments and successfully identified >100 peaks of PLs determined by parent ion m/z value. Further tandem mass spectrometric analysis (MS/MS) was performed to those PLs that are anatomically important according to their distribution on rat brain tissue section.

Direct tissue imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization and time-of-flight (MALDI-TOF) mass spectrometry has become increasingly important in biology and medicine, because this technology can detect the relative abundance and spatial distribution of interesting proteins in tissues. Five thyroid cancer samples, along with normal tissue, were sliced and transferred onto conductive glass slides. After laser scanning by MALDI-TOF equipped with a smart beam laser, images were created for individual masses and proteins were classified at 200-µm spatial resolution. Based on the spatial distribution, region-specific proteins on a tumor lesion could be identified by protein extraction from tumor tissue and analysis using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Using all the spectral data at each spot, various intensities of a specific peak were detected in the tumor and normal regions of the thyroid. Differences in the molecular weights of expressed proteins between tumor and normal regions were analyzed using unsupervised and supervised clustering. To verify the presence of discovered proteins through IMS, we identified ribosomal protein P2, which is specific for cancer. We have demonstrated the feasibility of IMS as a useful tool for the analysis of tissue sections, and identified the tumor-specific protein ribosomal protein P2. Graphical Abstract Keywords: Pathology, Proteins, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Thyroid Gland, Neoplasms INTRODUCTION Papillary thyroid carcinomas (PTC) are the most common form of thyroid cancer, accounting for nearly 85% of primary thyroid malignancies. Commonly, the solitary nodule is a palpably discrete swelling within an otherwise apparently normal thyroid gland, and is incidentally detected by radiological evaluation (1). From a clinical standpoint, the possibility of neoplastic disease is a major cause for concern in patients who present with thyroid nodules, because most conventional papillary carcinomas present as asymptomatic thyroid nodules. Recent studies have demonstrated 2 molecular mechanisms that function in the carcinogenesis of thyroid cancer: the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/Akt pathways (2, 3). Additionally, such pathways are related to genetic alterations such as rearrangements in the "rearranged during transfection" (RET) or neurotrophic tyrosine kinase receptor 1 (NTRK1) genes, and the activation of point mutations in the BRAF gene (4). In a previous study, protein expression patterns according to genetic variables have been demonstrated to correlate with the microscopic features, clinical manifestations, and prognostic characteristics of PTC (5). Although various protein alterations have been detected as diagnostic markers or prognostic predictors, the challenge remains to identify the expression of new molecules in tumors. In biological research, protein expression profiling technology is a useful method to identify differential protein expression patterns and modifications. Analytical techniques with high sensitivity and increased throughput are required for the discovery of new biomarkers and new drugs. Recent progress in imaging mass spectrometry (IMS) has made it possible to identify several cellular components such as proteins, drugs, and other endogenous molecules directly on tissue sections (6, 7, 8, 9, 10). IMS uses matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) technology. The matrix is applied on cryosectioned tissue and a very small area of the matrix-applied tissue is analyzed using MALDI-MS. Differences between the analyzed areas are displayed by the imaging program. Each analyzed area of the tissue section, which can be as small as >200 µm, on a conductive surface such as gold-plated or indium tin oxide (ITO)-coated slide, is analyzed spot by spot using MALDI-MS after the application of the matrix. Using all the spectral data from each spot, the magnitudes of specific peaks on each spot can be displayed in terms of color intensity. In this way, spatial information on the tissue can be obtained. Based on the spatial distribution, region-specific proteins on a tissue can be identified by extracting proteins from the tissue, digesting them with trypsin, and analyzing the fragments using liquid chromatography with tandem MS (LC-MS/MS). This technology using IMS has broad applications in the detection of new proteins in various fields. Compared with conventional imaging methods, the advantages of IMS are that it does not require the additional use of a specific antibody against the protein, and that it integrates histopathology and protein expression (11). For this reason, IMS provides superior information regarding distinct molecular arrangements in tissue sections. In the present study, we attempted to analyze thyroid samples, including those from PTC, using IMS. Before analysis using IMS, the diagnosis was confirmed by microscopic evaluation, immunohistochemical (IHC) staining of markers such as CK19, galectin-3, and RET, and pyrosequencing of the BRAF mutation, and then 5 homogenous samples of PTC were selected. After comparison of the differential molecular weight distribution between normal and tumor tissues using IMS, a protein of a specific molecular weight was identified by sequencing combined with LC-MS/MS, and then the presence of this protein was reconfirmed by western blot analysis.

Since the development of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, this procedure has been specifically used for analyzing proteins or high molecular weight compounds because of the interference of matrix signals in the regions of the low mass range. Recently, scientists have been using a wide range of chemical compounds as matrices that ionize small molecules in a mass spectrometer and overcome the limitations of MALDI mass spectrometry. In this study, we developed a new combination matrix of 3-hydroxycoumarin (3-HC) and 6-aza-2-thiothymine (ATT), which is capable of ionizing small molecules, including drugs and single amino acids. In addition to ionization of small molecules, the combination matrix by itself gives less signals in the low mass region and can be used for performing imaging mass spectrometry (IMS) experiments on tissues, which confirms the vacuum stability of the matrix inside a MALDI chamber. The drug donepezil was mapped in the intact tissue slices of mice simultaneously with a spatial resolution of 150 μm during IMS. IMS analysis clearly showed that intact donepezil was concentrated in the cortical region of the brain at 60 min after oral administration. Our observations and results indicate that the new combination matrix can be used for analyzing small molecules in complex samples using MALDI mass spectrometry.

Phospholipids are key components of cellular membrane and signaling. Among cellular phospholipids, phosphoinositides, phosphorylated derivatives of phosphatidylinositol are important as a participant in essential metabolic processes in animals. However, due to its low abundance in cells and tissues, it is difficult to identify the composition of phosphoinositides. Recent advances in mass spectrometric techniques, combined with established separation methods, have allowed the rapid and sensitive detection and quantification of a variety of lipid species including phosphoinositides. In this mini review, we briefly introduce progress in profiling of cellular phosphoinositides using mass spectrometry. We also summarize current progress of matrices development for the analysis of cellular phospholipids using matrix-assisted laser desorption/ionization mass spectrometry. The phosphoinositides profiling and phospholipids imaging will help us to understand how they function in a biological system and will provide a powerful tool for elucidating the mechanism of diseases such as diabetes, cancer and neurodegenerative diseases. The investigation of cellular phospholipids including phosphoinositides using electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry will suggest new insights on human diseases, and on clinical application through drug development of lipid related diseases.