Global changes of phospholipids identified by MALDI imaging mass spectrometry in a mouse model of Alzheimer's disease.

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 ß-amyloid (Aß). Disrupted Aß 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 Aß-42 (1). Our data showed changes in lipid distribution in the mouse frontal cortex, hippocampus, and subiculum, where Aß 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.

Imaging mass spectrometry in papillary thyroid carcinoma for the identification and validation of biomarker proteins.

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.

Disrupting TSLP-TSLP receptor interactions via putative small molecule inhibitors yields a novel and efficient treatment option for atopic diseases.

Thymic stromal lymphopoietin (TSLP) is a key player in atopic diseases, which has sparked great interest in therapeutically targeting TSLP. Yet, no small-molecule TSLP inhibitors exist due to the challenges of disrupting the protein-protein interaction between TSLP and its receptor. Here, we report the development of small-molecule TSLP receptor inhibitors using virtual screening and docking of >1,000,000 compounds followed by iterative chemical synthesis. BP79 emerged as our lead compound that effectively abrogates TSLP-triggered cytokines at low micromolar concentrations. For in-depth analysis, we developed a human atopic disease drug discovery platform using multi-organ chips. Here, topical application of BP79 onto atopic skin models that were co-cultivated with lung models and Th2 cells effectively suppressed immune cell infiltration and IL-13, IL-4, TSLP, and periostin secretion, while upregulating skin barrier proteins. RNA-Seq analysis corroborate these findings and indicate protective downstream effects on the lungs. To the best of our knowledge, this represents the first report of a potent putative small molecule TSLPR inhibitor which has the potential to expand the therapeutic and preventive options in atopic diseases.

Lipid Nanoparticle-Mediated Hit-and-Run Approaches Yield Efficient and Safe In Situ Gene Editing in Human Skin.

Despite exciting advances in gene editing, the efficient delivery of genetic tools to extrahepatic tissues remains challenging. This holds particularly true for the skin, which poses a highly restrictive delivery barrier. In this study, we ran a head-to-head comparison between Cas9 mRNA or ribonucleoprotein (RNP)-loaded lipid nanoparticles (LNPs) to deliver gene editing tools into epidermal layers of human skin, aiming for in situ gene editing. We observed distinct LNP composition and cell-specific effects such as an extended presence of RNP in slow-cycling epithelial cells for up to 72 h. While obtaining similar gene editing rates using Cas9 RNP and mRNA with MC3-based LNPs (10-16%), mRNA-loaded LNPs proved to be more cytotoxic. Interestingly, ionizable lipids with a pKa ∼ 7.1 yielded superior gene editing rates (55%-72%) in two-dimensional (2D) epithelial cells while no single guide RNA-dependent off-target effects were detectable. Unexpectedly, these high 2D editing efficacies did not translate to actual skin tissue where overall gene editing rates between 5%-12% were achieved after a single application and irrespective of the LNP composition. Finally, we successfully base-corrected a disease-causing mutation with an efficacy of ∼5% in autosomal recessive congenital ichthyosis patient cells, showcasing the potential of this strategy for the treatment of monogenic skin diseases. Taken together, this study demonstrates the feasibility of an in situ correction of disease-causing mutations in the skin that could provide effective treatment and potentially even a cure for rare, monogenic, and common skin diseases.

Global changes in phospholipids identified by MALDI MS in rats with focal cerebral ischemia.

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.

A new combination MALDI matrix for small molecule analysis: application to imaging mass spectrometry for drugs and metabolites.

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.

Binary matrix for MALDI imaging mass spectrometry of phospholipids in both ion modes.

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.

Identification of Protein Markers Specific for Papillary Renal Cell Carcinoma Using Imaging Mass Spectrometry.

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.

Lipid MALDI profile classifies non-small cell lung cancers according to the histologic type.

We investigated whether direct tissue matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) analysis on lipid may assist with the histopathologic diagnosis of non-small cell lung cancers (NSCLCs). Twenty-one pairs of frozen, resected NSCLCs and adjacent normal tissue samples were initially analyzed using histology-directed, MALDI MS. 2,5-dihydroxybenzoic acid/α-cyano-4-hydroxycinnamic acid were manually deposited on areas of each tissue section enriched in epithelial cells to identify lipid profiles, and mass spectra were acquired using a MALDI-time of flight instrument. A lipid profile that could differentiate cancer and adjacent normal samples with a median accuracy of 92.9% was discovered. Several phospholipids including phosphatidylcholines (PC) {34:1} were overexpressed in lung cancer. Squamous cell carcinomas and adenocarcinomas were found to have different lipid profiles. Discriminatory lipids correctly classified the histology of 80.4% of independent NSCLC surgical tissue samples (41 out of 51) in validation set. MALDI MS image of 11 discriminatory lipids validated their differential expression according to the histologic type in cancer cells of bronchoscopic biopsy samples. PC {32:0} [M+Na](+) (m/z 756.68) and ST-OH {42:1} [M-H](-) (m/z 906.89) were overexpressed in adenocarcinomas. Thus, lipid profiles accurately distinguish tumor from adjacent normal tissue and classify non-small cell lung cancers according to the histologic type.

Mass spectrometry based cellular phosphoinositides profiling and phospholipid analysis: a brief review.

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.