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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.

This article is available online at http://www.jlr.org Several hypotheses regarding the cause of AD have been proposed, among which the amyloid hypothesis is the most widely accepted ( 2 ). According to this hypothesis, AD is caused by abnormal accumulation of misfolded ␤ -amyloid (A ␤ ), a sequential cleavage product produced from amyloid precursor protein (APP) by ␤ -and ␥ -secretases, and hyperphosphorylated tau protein. Several lines of evidence support the idea that A ␤ can trigger the hyperphosphorylation of tau, resulting in neuronal degeneration in the brain ( 3, 4 ).There is evidence that FFAs are associated with several signaling processes related to the pathogenesis of AD. In particular, palmitic and stearic acid induce AD by triggering hyperphosphorylation of tau protein ( 5 ). Membrane phospholipids (PLs) are a prominent source of FFAs. Under disease conditions, phospholipase enzymes are produced in excess, inducing the hydrolysis of PLs and the release of FFAs. It is reasonable to hypothesize that alterations in PL concentrations are closely related to the pathogenesis of AD, as PLs perform a number of important cellular roles, including stabilization of membrane ion channels, neurotransmission, and the localization of A ␤ plaques to PL cores ( 6, 7 ).As a result of progress in MS, and MALDI-imaging MS (IMS) in particular, the analysis of PL changes during disease progression has become feasible. MALDI-IMS is a unique tool that integrates molecular and histological information together with information attained using Alzheimer's disease (AD), the most common form of dementia, is primarily caused by abnormal protein kinesis and the accumulation of aggregated proteins in the brain. This work was supported

has been given to cellular phospholipid analyses compared with protein or peptide analyses. However, recent progress in mass spectrometric analysis has enabled the identifi cation of small cellular components including phospholipids and quantifi cation of their cellular expression profi les ( 3, 4 ).ESI MS is widely used for phospholipid analysis. Currently, MALDI imaging MS (MALDI IMS) has become the prime choice because it provides additional advantages over ESI, including simple sample preparation and an abundance of information about molecular structures and spatial data ( 3,(5)(6)(7)(8)(9). The principal advantage of MALDI IMS is its ability to ionize molecules directly from the surface of tissue samples, which can demonstrate the spatial distribution of molecules of interest. However, MALDI IMS must be further improved to resolve technical problems such as the ambiguity of structural determination, unequal ionization effi ciencies of different compounds, and complications due to uneven tissue structure. Glycerophospholipids can be classifi ed according to their head groups, linkage between the polar chain and glycerol backbone, and variability in their FA composition ( 9 ). Depending on the presence of amine or hydroxyl structures on their head group, phospholipids can be ionized in either positive or negative mode. The ionization effi ciency of lipids can also be affected by their head group structure. Occasionally, it is diffi cult to perform MALDI IMS analysis twice in both positive and negative ionization modes on the same tissue sample to identify lipid composition Abstract 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 confi rmed 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 identifi cation of phospholipid markers for ischemic injury and successfully identifi ed several distinctly located phospholipids in ischemic brain tissue.

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.

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.

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