MALDI Mass Spectrometry Research Articles

MALDI target functionalization with deposited thin films of lanthanum stearate – An efficient tool for in situ enrichment of human globin adducts of chlorinated organic compounds

“Lab-on-a-plate” methodology encapsulates a number of different approaches aimed to shorten extensive sample processing for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Lately, we have proposed two approaches to the enrichment of chlorine-containing adducts of human globin (HHb) in a “lab-on-a-plate” format based on different techniques for the sorbent application to MALDI target spots: (i) droplet-free electrospraying of metal oxide nanoparticles and (ii) deposition of lanthanum stearate thin films (FLa). The latter approach has turned out to be more time- and cost-effective and, therefore, seems to be more promising. In this work, we assessed the quantitative characteristics (sensitivity, selectivity) of the “thin-film” procedure in order to estimate its analytical potential. The FLa-modified MALDI target was used for the on-spot enrichment of HHb adducts from the tryptic digests of the protein incubated with two model mono- and dichlorine-containing alkylating agents. The sensitivity of the technique is in the range of hundreds of fmol. Furthermore, the FLa-based procedure shows high selectivity (maximum molar ratio of modified/unmodified form of LLGNVLVC112VLAHHFGK is 1:500). As a case study, on-plate extraction of HHb adducts, formed due to the interaction of the protein with in vitro generated diclofenac metabolites, was performed on FLa-functionalized spots. This resulted in specific extraction of the HHb adducts with two diclofenac oxidation products from the tryptic digest of modified HHb. The developed approach appears to be effective for in situ enrichment of chlorine-containing adducts of HHb.

What Are the Key Factors for the Detection of Peptides Using Mass Spectrometry on Boron-Doped Diamond Surfaces?

We investigated the use of boron-doped diamond (BDD) with different surface morphologies for the enhanced detection of nine different peptides by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). For the first time, we compared three different nanostructured BDD film morphologies (Continuous, Nanograss, and Nanotips) with differently terminated surfaces (-H, -O, and -F) to commercially available Ground Steel plates. All these surfaces were evaluated for their effectiveness in detecting the nine different peptides by MALDI-MS. Our results demonstrated that certain nanostructured BDD surfaces exhibited superior performance for the detection of especially hydrophobic peptides (e.g., bradykinin 1-7, substance P, and the renin substrate), with a limit of detection of down to 2.3 pM. Further investigation showed that hydrophobic peptides (e.g., bradykinin 1-7, substance P, and the renin substrate) were effectively detected on hydrogen-terminated BDD surfaces. On the other hand, the highly acidic negatively charged peptide adrenocorticotropic hormone fragment 18-39 was effectively identified on oxygen-/fluorine-terminated BDD surfaces. Furthermore, BDD surfaces reduced sodium adduct contamination significantly.

Study on the Anti-Inflammatory Mechanism of Coumarins in Peucedanum decursivum Based on Spatial Metabolomics Combined with Network Pharmacology.

Peucedanum decursivum (Miq.) Maxim (P. decursivum) is a traditional Chinese medicinal plant with pharmacological effects such as anti-inflammatory and anti-tumor effects, the root of which is widely used as medicine. Determining the spatial distribution and pharmacological mechanisms of metabolites is necessary when studying the effective substances of medicinal plants. As a means of obtaining spatial distribution information of metabolites, mass spectrometry imaging has high sensitivity and allows for molecule visualization. In this study, matrix-assisted laser desorption mass spectrometry (MALDI-TOF-MSI) and network pharmacology were used for the first time to visually study the spatial distribution and anti-inflammatory mechanism of coumarins, which are metabolites of P. decursivum, to determine their tissue localization and mechanism of action. A total of 27 coumarins were identified by MALDI-TOF-MSI, which mainly concentrated in the cortex, periderm, and phloem of the root of P. decursivum. Network pharmacology studies have identified key targets for the anti-inflammatory effect of P. decursivum, such as TNF, PTGS2, and PRAKA. GO enrichment and KEGG pathway analyses indicated that coumarins in P. decursivum mainly participated in biological processes such as inflammatory response, positive regulation of protein kinase B signaling, chemical carcinogenesis receptor activation, pathways in cancer, and other biological pathways. The molecular docking results indicated that there was good binding between components and targets. This study provides a basis for understanding the spatial distribution and anti-inflammatory mechanism of coumarins in P. decursivum.

Direct and Rapid Visualization of the Spatial Distribution of Cholesterol in Alzheimer's and Cancer Tissue via MALDI Mass Spectrometry Imaging.

Cholesterol is a vital component of the central nervous system and tissues, and understanding its spatial distribution is crucial for biology, pathophysiology, and diagnostics. However, direct imaging of cholesterol using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) remains challenging and time-consuming due to the difficulty in ionizing the sterol molecule. To tackle this issue, a MALDI-MSI method is established for direct and rapid analysis of the spatial distribution of cholesterol in Alzheimer's disease (AD), different cancer tissues and organs via MALDI-MSI. This excellent imaging performance depends on the study and systemic optimization of various conditions that affect the imaging of MALDI-MSI. In this case, we report the distribution and levels of cholesterol across specific structures of the AD mouse brain and different tumor tissue and organs. According to the results, the content of cholesterol in the AD mouse cerebellum, especially in the arborvitae, was significantly higher than that in the wild type (WT) model. Furthermore, we successfully visualize the distribution of cholesterol in other organs, such as the heart, liver, spleen, kidney, pancreas, as well as tumor tissues parenchyma and interstitium using MALDI-MSI. Notably, the attribution of cholesterol MS/MS hydrocarbon fragments was systematically investigated. Our presented optimization strategy and established MALDI-MSI method can be easily generalized for different animal tissues or live samples, thereby facilitating the potential for applications of MALDI-MSI in clinical, medical and biological research.

Distribution of active ingredients and quality control of Forsythia suspensa with AP-MALDI mass spectrometry imaging.

The fruits of Forsythia suspensa (F. suspensa) have been used as a traditional Chinese medicine for 2000 years. Currently, the quality control of F. suspensa strictly follows the instructions of Chinese Pharmacopeia, which mainly controls the content of forsythoside A, phillyrin, and volatile oil. In this study, air pressure MALDI mass spectrometry imaging (AP-MALDI MSI) was used to evaluate the quality of F. suspensa fruits and the distribution of dozens of active ingredients. The variation of active ingredients was measured for more than 30 batches of samples, regarding harvest time, cultivated environment, shelf-life, and habitat. Fifty-three active ingredients could be detected in F. suspensa fruits with AP-MALDI MSI. Seven active ingredients were upregulated, four ingredients downregulated, and 15 ingredients did not change in ripe fruits. A sharp variation of active ingredients in late September was observed for the Caochuan fruits harvested in 2019, which is closely related to the appearance of the ginger color of the pericarp under the microscope observation. The microscope observation is a reliable way to classify ripe and green fruits instead of outlook. Just considering forsythoside A and phillyrin, it is found that wild fruits are better than cultivated fruits, but cultivated fruits have high contents of other ingredients. The shelf-life of F. suspensa fruits is proposed to be 3 years, considering the 26 ingredients investigated. It was found that Luoning wild fruits are better than those from Caochuan with a new evaluation method. Mass spectrometry imaging is an easy, objective, and effective method to evaluate the quality of F. suspensa fruits.

MALDI Mass Spectrometry on High-Density Droplet Arrays: Matrix Deposition, Selective Removal, and Recrystallization.

High-density droplet arrays are emerging as a powerful tool for high-throughput bioanalytical applications. These arrays are formed of thousands of nanoliter droplets, which can be analyzed by various optical and spectroscopic methods as well as label-free matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). However, special precautions are required for the massive number of small droplets, particularly in the deposition of matrix compounds. Here, we introduce a new workflow for the analytical preparation of an array comprising 6048 droplets, which significantly improves the intensity of the MALDI-MS signals. We deposited matrix compounds in a custom-made sublimation chamber followed by a recrystallization step to achieve significant signal intensity increases for three model proteins with low, medium, and large masses, respectively. Furthermore, selective removal of the matrix before recrystallization enhanced the spatial resolution and increased the signal intensity by an average of 57%. This method can be easily standardized and upscaled for the preparation of an even larger number of droplets per array for MS analysis.

Effect of dynamic exclusion and the use of FAIMS, DIA and MALDI-mass spectrometry imaging with ion mobility on amyloid protein identification

Amyloidosis is a disease characterized by local and systemic extracellular deposition of amyloid protein fibrils where its excessive accumulation in tissues and resistance to degradation can lead to organ failure. Diagnosis is challenging because of approximately 36 different amyloid protein subtypes. Imaging methods like immunohistochemistry and the use of Congo red staining of amyloid proteins for laser capture microdissection combined with liquid chromatography tandem mass spectrometry (LMD/LC–MS/MS) are two diagnostic methods currently used depending on the expertise of the pathology laboratory. Here, we demonstrate a streamlined in situ amyloid peptide spatial mapping by Matrix Assisted Laser Desorption Ionization–Mass Spectrometry Imaging (MALDI-MSI) combined with Trapped Ion Mobility Spectrometry for potential transthyretin (ATTR) amyloidosis subtyping. While we utilized the standard LMD/LC–MS/MS workflow for amyloid subtyping of 31 specimens from different organs, we also evaluated the potential introduction in the MS workflow variations in data acquisition parameters like dynamic exclusion, or testing Data Dependent Acquisition combined with High-Field Asymmetric Waveform Ion Mobility Spectrometry (DDA FAIMS) versus Data Independent Acquisition (DIA) for enhanced amyloid protein identification at shorter acquisition times. We also demonstrate the use of Mascot’s Error Tolerant Search and PEAKS de novo sequencing for the sequence variant analysis of amyloidosis specimens.

Visualization of renal rotenone accumulation after oral administration and in situ detection of kidney injury biomarkers via MALDI mass spectrometry imaging.

The examination of drug accumulation within complex biological systems offers valuable insights into the molecular aspects of drug metabolism and toxicity. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is an innovative methodology that enables the spatial visualization and quantification of biomolecules as well as drug and its metabolites in complex biological system. Hence, this method provides valuable insights into the metabolic profile and any molecular changes that may occur as a result of drug treatment. The renal system is particularly vulnerable to adverse effects of drug-induced harm and toxicity. In this study, MALDI MSI was utilized to examine the spatial distribution of drug and renal metabolites within kidney tissues subsequent to a single oral dosage of the anticancer compound rotenone. The integration of ion mobility spectrometry with MALDI MSI enhanced the data acquisition and analysis, resulting to improved mass resolution. Subsequently, the MS/MS fragment ions of rotenone reference drug were detected and characterized using MALDI HDMS/MS imaging. Notably, drug accumulation was observed in the cortical region of the representative kidney tissue sections treated with rotenone. The histological examination of treated kidney tissues did not reveal any observable changes. Differential ion intensity of renal endogenous metabolites was observed between untreated and rotenone-treated tissues. In the context of treated kidney tissues, the ion intensity level of sphingomyelin (D18:1/16:0), a sphingolipid indicator of glomerular cell injury and renal damage, was found to be elevated significantly compared to untreated kidney tissues. Conversely, the ion intensities of choline, glycero-3-phosphocholine (GPC), inosine, and a lysophosphatidylcholine LysoPC(18:0) exhibited a significant decrease. The results of this study demonstrate the potential of MALDI MSI as a novel technique for investigating the in situ spatial distribution of drugs and renal endogenous molecules while preserving the anatomical integrity of the kidney tissue. This technique can be used to study drug-induced metabolism and toxicity in a dynamic manner.

Lipid signature associated with chronic colon inflammation reveals a dysregulation in colonocyte differentiation process

Inflammatory Bowel Disease (IBD) comprises a heterogeneous group of chronic inflammatory conditions of the gastrointestinal tract that include ulcerative colitis (UC) and Crohn's disease. Although the etiology is not well understood, IBD is characterized by a loss of the normal epithelium homeostasis that disrupts the intestinal barrier of these patients. Previous work by our group demonstrated that epithelial homeostasis along the colonic crypts involves a tight regulation of lipid profiles. To evaluate whether lipidomic profiles conveyed the functional alterations observed in the colonic epithelium of IBD, we performed matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) analyses of endoscopic biopsies from inflamed and non-inflamed segments obtained from UC patients. Our results indicated that lipid profiling of epithelial cells discriminated between healthy and UC patients. We also demonstrated that epithelial cells of the inflamed mucosa were characterized by a decrease in mono- and di-unsaturated fatty acid-containing phospholipids and higher levels of arachidonic acid-containing species, suggesting an alteration of the lipid gradients occurring concomitantly to the epithelial differentiation. This result was reinforced by the immunofluorescence analysis of EPHB2 and HPGD, markers of epithelial cell differentiation, sustaining that altered lipid profiles were at least partially due to a faulty differentiation process. Overall, our results showed that lipid profiling by MALDI-MSI faithfully conveys molecular and functional alterations associated with the inflamed epithelium, providing the foundation for a novel molecular characterization of UC patients.

Quantitative Analysis of Blended Oils Based on Intensity Ratios of Marker Ions in MALDI-MS Spectra.

Determination of quantitative compositions of blended oils is an essential but challenging step for the quality control and safety assurance of blended oils. We herein report a method for the quantitative analysis of blended oils based on the intensity ratio of triacylglycerol marker ions, which could be obtained from the highly reproducible spectra acquired by using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to directly analyze blended oils in their oily states. We demonstrated that this method could provide good quantitative results to binary, ternary, and quaternary blended oils, with simultaneous quantitation of multiple compositions, and was applicable for quantitative analysis of commercial blended oil products. Moreover, the intensity ratio-based method could be used to rapidly measure the proportions of oil compositions in blended oils, only based on the spectra of the blended oils and related pure oils, making the method as a high-throughput approach to meet the sharply growing analytical demands of blended oils.

Characterising fragmentation of compostable bioplastic: releasing microplastics or small bioplastic debris

BackgroundPlastic is generating global pollution and the replacement such as bioplastic has been developed to mitigate the pollution. To this end, the fate, transformation and pathway of bioplastics need more research. For example, the fragmentation of bioplastic can release small debris that can be categorised as microplastics, which is tested herein by taking an example of a compostable plastic that is used as a bin bag on our kitchen table to collect the food residues.ResultsFirst, we employ matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) to identify the main components of the bioplastic bag as polymer and starch. Next, we use Raman imaging to monitor the stability under laser illumination, in an oven at ~ 60 °C for ~ 2 weeks, or in the presence of tap water for half a year. Basically, the compostable plastic is stable under these conditions. Thirdly, however, once used as table-bin bag with involvement of food residues, within ~ 1 week, the bioplastic bag is broken and biodegraded to release debris. The derivate surface groups are effectively monitored and directly visualised via Raman imaging, and cross-checked with scanning electron microscope (SEM). The yielded small molecule such as formic acid is also identified, along with the released debris of microplastics, with the help of on-site extraction of the fragmented sample and imaging analysis algorithm of the hyper spectrum.ConclusionsAfter one week, the bag in the waste bin fragments, releasing a significant amount of debris. This could pose a functional issue if users intend to use the bag for at least a week, and could become a potential environmental problem if the waste is dispersed uncontrollably. In general, further research is needed to potentially distinguish the persistent conventional microplastics from the bioplastic fragments, to effectively mitigate the plastic pollution.Graphical

Mass Spectrometry Imaging of Coniine and Other Hemlock Alkaloids after On-Tissue Derivatization Reveals Distinct Alkaloid Distributions in the Plant.

Specialized metabolites play important roles in plants and can, for example, protect plants from predators or pathogens. Alkaloids, due to their pronounced biological activity on higher animals, are one of the most intriguing groups of specialized metabolites, and many of them are known as plant defense compounds. Poison hemlock, Conium maculatum, is well-known for its high content of piperidine alkaloids, of which coniine is the most famous. The distribution, localization, and diversity of these compounds in C. maculatum tissues have not yet been studied in detail. The hemlock alkaloids are low molecular weight compounds with relatively high volatility. They are thus difficult to analyze on-tissue by MALDI mass spectrometry imaging due to delocalization, which occurs even when using an atmospheric pressure ion source. In this manuscript, we describe an on-tissue derivatization method that allows the subsequent determination of the spatial distribution of hemlock alkaloids in different plant tissues by mass spectrometry imaging. Coniferyl aldehyde was found to be a suitable reagent for derivatization of the secondary amine alkaloids. The imaging analysis revealed that even chemically closely related hemlock alkaloids are discretely distributed in different plant tissues. Additionally, we detected a yet undescribed hemlock alkaloid in Conium maculatum seeds.

Application of a robust MALDI mass spectrometry approach for bee pollen investigation

Pollen collected by pollinators can be used as a marker of the foraging behavior as well as indicate the botanical species present in each environment. Pollen intake is essential for pollinators’ health and survival. During the foraging activity, some pollinators, such as honeybees, manipulate the collected pollen mixing it with salivary secretions and nectar (corbicular pollen) changing the pollen chemical profile. Different tools have been developed for the identification of the botanical origin of pollen, based on microscopy, spectrometry, or molecular markers. However, up to date, corbicular pollen has never been investigated. In our work, corbicular pollen from 5 regions with different climate conditions was collected during spring. Pollens were identified with microscopy-based techniques, and then analyzed in MALDI-MS. Four different chemical extraction solutions and two physical disruption methods were tested to achieve a MALDI-MS effective protocol. The best performance was obtained using a sonication disruption method after extraction with acetic acid or trifluoroacetic acid. Therefore, we propose a new rapid and reliable methodology for the identification of the botanical origin of the corbicular pollens using MALDI-MS. This new approach opens to a wide range of environmental studies spanning from plant biodiversity to ecosystem trophic interactions.Graphical

Graphite Sheet-Assisted Laser Desorption Ionization-Mass Spectrometry for Small Organic Compound Analysis.

Carbon-based nanopowders have been used as ionization materials for laser desorption ionization-mass spectrometry (LDI-MS) and are very efficient at detection in low m/z regions. In this study, we aimed to develop a new sheet-type graphite material that possessed a randomly grooved nanostructured surface consisting of developed sp2-conjugated atomic carbon to facilitate the desorption/ionization of small compounds in LDI-MS. The graphite sheet exhibited higher UV absorption and provided higher ionization efficiency and survival yield in the LDI-MS detection of a thermometer ion, 4-chloro-benzopyridinium, than those of highly oriented graphite plates. These properties demonstrate that the present graphite sheet is suited for use as an LDI-MS material. Graphite sheet-assisted LDI-MS successfully detected various substances, including amino acids, peptides, and polyethylene glycol polymers, with higher ion intensities and less noise than those associated with conventional organic matrix-assisted LDI-MS (MALDI-MS). Furthermore, graphite sheet-assisted LDI-MS analysis provided more peaks (252 peaks) derived from soy sauce than those obtained by MALDI-MS (36 peaks) and required fewer preparation processes (dilution and air-dried) compared with previously established graphite carbon black-assisted LDI-MS (171 peaks) in the positive mode. This study demonstrates that graphite sheet-assisted LDI-MS has the potential for small organic compound analyses in the biomedical and food science fields.

Large-Scale Screening of Pharmaceutical Compounds to Explore the Application Space of On-Tissue MALDI and MALDI-2 Mass Spectrometry.

The successful application of matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) in pharmaceutical research is strongly dependent on the detection of the drug of interest at physiologically relevant concentrations. Here we explored how insufficient sensitivity due to low ionization efficiency and/or the interaction of the drug molecule with the local biochemical environment of the tissue can be mitigated for many compound classes using the recently introduced MALDI-MSI coupled with laser-induced postionization, known as MALDI-2-MSI. Leveraging a MALDI-MSI screen of about 1,200 medicines/drug-like compounds from a broad range of medicinal application areas, we demonstrate a significant improvement in drug detection and the degree of sensitivity uplift by using MALDI-2 versus traditional MALDI. Our evaluation was made under simulated imaging conditions using liver homogenate sections as substrate, onto which the compounds were spotted to mimic biological conditions to the first order. To enable an evaluable detection by both MALDI and MALDI-2 for the majority of employed compounds, we spotted 1 μL of a 10 mM solution using a spotting robot and performed our experiments with a Bruker timsTOF fleX MALDI-2 instrument in both positive and negative ion modes. Specifically, we demonstrate using a large cohort of drug-like compounds that ∼60% of the tested compounds showed a more than 10-fold increase in signal intensity and ∼16% showed a more than 100-fold increase upon use of MALDI-2 postionization. Such increases in sensitivity could help advance pharmaceutical MALDI-MSI applications toward the single-cell level.

A Simple and Rapid Microscale Method for Isolating Bacterial Lipopolysaccharides.

Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.

Differential distribution of characteristic constituents in peel and pulp of Aurantii Fructus Immaturus (Citrus aurantium L.) using MALDI mass spectrometry imaging

Aurantii Fructus Immaturus (AFI) was structurally divided into two parts named “peel” and “pulp”. The exocarp and mesocarp of materials named “peel”. The endocarp separated into multiple compartments and the cystic hair attached to it named “pulp”. In order to explore the distribution and content of constituents in AFI, an efficient method to explore the distribution of constituents was developed based on matrix assisted laser desorption/ionization fourier transform ion cyclotron resonance mass spectrometry imaging (MALDI-FTICR-MSI). After simple processing, thirty-two constituents with distinct localization in the mass range of 101–1200 Da were identified by MALDI-FTICR-MSI. In addition, the identified four flavnoids (poncirin, sinensetin, 3,5,6,7,8,3′,4′-heptemthoxyflavone, and tangeritin) were analyzed for differences between using LC-MS. Quantitative analysis results supported the quantitative results from MALDI-FT-ICR-MSI. The results implied that different parts had different constituents in AFI, and demonstrated MALDI-MSI have high potential in the direct analysis of constituents.

Chemical signatures delineate heterogeneous amyloid plaque populations across the Alzheimer's disease spectrum.

Amyloid plaque deposition is recognized as the primary pathological hallmark of Alzheimer's disease(AD) that precedes other pathological events and cognitive symptoms. Plaque pathology represents itself with an immense polymorphic variety comprising plaques with different stages of amyloid fibrillization ranging from diffuse to fibrillar, mature plaques. The association of polymorphic Aβ plaque pathology with AD pathogenesis, clinical symptoms and disease progression remains unclear. Advanced chemical imaging tools, such as functional amyloid microscopy combined with MALDI mass spectrometry imaging (MSI), are now enhanced by deep learning algorithms. This integration allows for precise delineation of polymorphic plaque structures and detailed identification of their associated Aβ compositions. We here set out to make use of these tools to interrogate heterogenic plaque types and their associated biochemical architecture. Our findings reveal distinct Aβ signatures that differentiate diffuse plaques from fibrilized ones, with the latter showing substantially higher levels of Aβx-40. Notably, within the fibrilized category, we identified a distinct subtype known as coarse-grain plaques. Both in sAD and fAD brain tissue, coarse grain plaques contained more Aβx-40 and less Aβx-42 compared with cored plaques. The coarse grain plaques in both sAD and fAD also showed higher levels of neuritic content including paired helical filaments (PHF-1)/phosphorylated phospho Tau-immunopositive neurites. Finally, the Aβ peptide content in coarse grain plaques resembled that of vascular Aβ deposits (CAA) though with relatively higher levels of Aβ1-42 and pyroglutamated Aβx-40 and Aβx-42 species in coarse grain plaques. This is the first of its kind study on spatial in situ biochemical characterization of different plaque morphotypes demonstrating the potential of the correlative imaging techniques used that further increase the understanding of heterogeneous AD pathology. Linking the biochemical characteristics of amyloid plaque polymorphisms with various AD etiologies and toxicity mechanisms is crucial. Understanding the connection between plaque structure and disease pathogenesis can enhance our insights. This knowledge is particularly valuable for developing and advancing novel, amyloid-targeting therapeutics.

EDI3 knockdown in ER-HER2+ breast cancer cells reduces tumor burden and improves survival in two mouse models of experimental metastasis

BackgroundDespite progress understanding the mechanisms underlying tumor spread, metastasis remains a clinical challenge. We identified the choline-producing glycerophosphodiesterase, EDI3 and reported its association with metastasis-free survival in endometrial cancer. We also observed that silencing EDI3 slowed cell migration and other cancer-relevant phenotypes in vitro. Recent work demonstrated high EDI3 expression in ER-HER2+ breast cancer compared to the other molecular subtypes. Silencing EDI3 in ER-HER2+ cells significantly reduced cell survival in vitro and decreased tumor growth in vivo. However, a role for EDI3 in tumor metastasis in this breast cancer subtype was not explored. Therefore, in the present work we investigate whether silencing EDI3 in ER-HER2+ breast cancer cell lines alters phenotypes linked to metastasis in vitro, and metastasis formation in vivo using mouse models of experimental metastasis.MethodsTo inducibly silence EDI3, luciferase-expressing HCC1954 cells were transduced with lentiviral particles containing shRNA oligos targeting EDI3 under the control of doxycycline. The effect on cell migration, adhesion, colony formation and anoikis was determined in vitro, and significant findings were confirmed in a second ER-HER2+ cell line, SUM190PT. Doxycycline-induced HCC1954-luc shEDI3 cells were injected into the tail vein or peritoneum of immunodeficient mice to generate lung and peritoneal metastases, respectively and monitored using non-invasive bioluminescence imaging. Metabolite levels in cells and tumor tissue were analyzed using targeted mass spectrometry and MALDI mass spectrometry imaging (MALDI-MSI), respectively.ResultsInducibly silencing EDI3 reduced cell adhesion and colony formation, as well as increased susceptibility to anoikis in HCC1954-luc cells, which was confirmed in SUM190PT cells. No influence on cell migration was observed. Reduced luminescence was seen in lungs and peritoneum of mice injected with cells expressing less EDI3 after tail vein and intraperitoneal injection, respectively, indicative of reduced metastasis. Importantly, mice injected with EDI3-silenced cells survived longer. Closer analysis of the peritoneal organs revealed that silencing EDI3 had no effect on metastatic organotropism but instead reduced metastatic burden. Finally, metabolic analyses revealed significant changes in choline and glycerophospholipid metabolites in cells and in pancreatic metastases in vivo.ConclusionsReduced metastasis upon silencing supports EDI3’s potential as a treatment target in metastasizing ER-HER2+ breast cancer.

Development of a New Binary Matrix for the Comprehensive Analysis of Lipids and Pigments in Micro- and Macroalgae Using MALDI-ToF/ToF Mass Spectrometry.

While edible algae might seem low in fat, the lipids they contain are crucial for good health and preventing chronic diseases. This study introduces a binary matrix to analyze all the polar lipids in both macroalgae (Wakame-Undaria pinnatifida, Dulse-Palmaria palmata, and Nori-Porphyra spp.) and microalgae (Spirulina-Arthrospira platensis, and Chlorella-Chlorella vulgaris) using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The key lies in a new dual matrix made by combining equimolar amounts of 1,5-diaminonaphthalene (DAN) and 9-aminoacridine (9AA). This combination solves the limitations of single matrices: 9AA is suitable for sulfur-containing lipids and acidic phospholipids, while DAN excels as an electron-transfer secondary reaction matrix for intact chlorophylls and their derivatives. By employing the equimolar binary matrix, a wider range of algal lipids, including free fatty acids, phospholipids, glycolipids, pigments, and even rare arsenosugarphospholipids were successfully detected, overcoming drawbacks related to ion suppression from readily ionizable lipids. The resulting mass spectra exhibited a good signal-to-noise ratio at a lower laser fluence and minimized background noise. This improvement stems from the binary matrix's ability to mitigate in-source decay effects, a phenomenon often encountered for certain matrices. Consequently, the data obtained are more reliable, facilitating a faster and more comprehensive exploration of algal lipidomes using high-throughput MALDI-MS/MS analysis.