Spatial Omics Reveals that Cancer-Associated Glycan Changes Occur Early in Liver Disease Development in a Western Diet Mouse Model of MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a progressive disease and comprises different stages of liver damage; it is significantly associated with obese and overweight patients. Untreated MASLD can progress to life-threatening end-stage conditions, such as cirrhosis and liver cancer. N-Linked glycosylation is one of the most common post-translational modifications in the cell surface and secreted proteins. N-Linked glycan alterations have been established to be signatures of liver diseases. However, the N-linked glycan changes during the progression of MASLD to liver cancer are still unknown. Here, we induced different stages of MASLD in mice and liver-cancer-related phenotypes and elucidated the N-glycome profile during the progression of MASLD by quantitative and qualitative profiling in situ using matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS). Importantly, we identified specific N-glycan structures including fucosylated and highly branched N-linked glycans at very early stages of liver injury (steatosis), which in humans are associated with cancer development, establishing the importance of these modifications with disease progression. Finally, we report that N-linked glycan alterations can be observed in our models by MALDI-IMS before liver injury is identified by histological analysis. Overall, we propose these findings as promising biomarkers for the early diagnosis of liver injury in MASLD.

Histochemistry and Cell Biology-a glance into the past and a look ahead.

At the occasion of the 65th anniversary of Histochemistry and Cell Biology, we browse through its first ten years of publication and highlight a selection of papers from the early days of enzyme, protein, and carbohydrate histochemistry. In addition, we narrate recent progress to identify, quantify, and precisely determine the tissue localization of proteins and lipids, and small molecules by the combination of spectroscopic techniques and histology.

High-Resolution Imaging Mass Spectrometry of Human Donor Eye: Photoreceptors Cells and Basal Laminar Deposit of Age-Related Macular Degeneration.

Pathologies of the retina are clinically visualized in vivo with OCT and ex vivo with immunohistochemistry. Although both techniques provide valuable information on prognosis and disease state, a comprehensive method for fully elucidating molecular constituents present in locations of interest is desirable. The purpose of this work was to use multimodal imaging technologies to localize the vast number of molecular species observed with matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) in aged and diseased retinal tissues. Herein, MALDI IMS was utilized to observe molecular species that reside in photoreceptor cells and also a basal laminar deposit from two human donor eyes. The molecular species observed to accumulate in these discrete regions can be further identified and studied to attempt to gain a greater understanding of biological processes occurring in debilitating eye diseases such as age-related macular degeneration (AMD).

MALDI imaging mass spectrometry: an emerging tool in neurology.

Neurological disease and disorders remain a large public health threat. Thus, research to improve early detection and/or develop more effective treatment approaches are necessary. Although there are many common techniques and imaging modalities utilized to study these diseases, existing approaches often require a label which can be costly and time consuming. Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is a label-free, innovative and emerging technique that produces 2D ion density maps representing the distribution of an analyte(s) across a tissue section in relation to tissue histopathology. One main advantage of MALDI IMS over other imaging modalities is its ability to determine the spatial distribution of hundreds of analytes within a single imaging run, without the need for a label or any a priori knowledge. Within the field of neurology and disease there have been several impactful studies in which MALDI IMS has been utilized to better understand the cellular pathology of the disease and or severity. Furthermore, MALDI IMS has made it possible to map specific classes of analytes to regions of the brain that otherwise may have been lost using more traditional methods. This review will highlight key studies that demonstrate the potential of this technology to elucidate previously unknown phenomenon in neurological disease.

Polyunsaturated Fatty Acid-Enriched Lipid Fingerprint of Glioblastoma Proliferative Regions Is Differentially Regulated According to Glioblastoma Molecular Subtype.

Glioblastoma (GBM) represents one of the deadliest tumors owing to a lack of effective treatments. The adverse outcomes are worsened by high rates of treatment discontinuation, caused by the severe side effects of temozolomide (TMZ), the reference treatment. Therefore, understanding TMZ's effects on GBM and healthy brain tissue could reveal new approaches to address chemotherapy side effects. In this context, we have previously demonstrated the membrane lipidome is highly cell type-specific and very sensitive to pathophysiological states. However, little remains known as to how membrane lipids participate in GBM onset and progression. Hence, we employed an ex vivo model to assess the impact of TMZ treatment on healthy and GBM lipidome, which was established through imaging mass spectrometry techniques. This approach revealed that bioactive lipid metabolic hubs (phosphatidylinositol and phosphatidylethanolamine plasmalogen species) were altered in healthy brain tissue treated with TMZ. To better understand these changes, we interrogated RNA expression and DNA methylation datasets of the Cancer Genome Atlas database. The results enabled GBM subtypes and patient survival to be linked with the expression of enzymes accounting for the observed lipidome, thus proving that exploring the lipid changes could reveal promising therapeutic approaches for GBM, and ways to ameliorate TMZ side effects.

Overview of how N32 and N34 elovanoids sustain sight by protecting retinal pigment epithelial cells and photoreceptors.

The essential fatty acid DHA (22:6, omega-3 or n-3) is enriched in and required for the membrane biogenesis and function of photoreceptor cells (PRCs), synapses, mitochondria, etc. of the CNS. PRC DHA becomes an acyl chain at the sn-2 of phosphatidylcholine, amounting to more than 50% of the PRC outer segment phospholipids, where phototransduction takes place. Very long chain PUFAs (n-3, ≥ 28 carbons) are at the sn-1 of this phosphatidylcholine molecular species and interact with rhodopsin. PRC shed their tips (DHA-rich membrane disks) daily, which in turn are phagocytized by the retinal pigment epithelium (RPE), where DHA is recycled back to PRC inner segments to be used for the biogenesis of new photoreceptor membranes. Here, we review the structures and stereochemistry of novel elovanoid (ELV)-N32 and ELV-N34 to be ELV-N32: (14Z,17Z,20R,21E,23E,25Z,27S,29Z)-20,27-dihydroxydo-triaconta-14,17,21,23,25,29-hexaenoic acid; ELV-N34: (16Z,19Z,22R,23E,25E,27Z,29S,31Z)-22,29-dihydroxytetra-triaconta-16,19,23,25,27,31-hexaenoic acid. ELVs are low-abundance, high-potency, protective mediators. Their bioactivity includes enhancing of antiapoptotic and prosurvival protein expression with concomitant downregulation of proapoptotic proteins when RPE is confronted with uncompensated oxidative stress. ELVs also target PRC/RPE senescence gene programming, the senescence secretory phenotype in the interphotoreceptor matrix, as well as inflammaging (chronic, sterile, low-grade inflammation). An important lesson on neuroprotection is highlighted by the ELV mediators that target the terminally differentiated PRC and RPE, sustaining a beautifully synchronized renewal process. The role of ELVs in PRC and RPE viability and function uncovers insights on disease mechanisms and the development of therapeutics for age-related macular degeneration, Alzheimer's disease, and other pathologies.

Familial Hyperaldosteronism Type 3 with a Rapidly Growing Adrenal Tumor: An In Situ Aldosterone Imaging Study.

Primary aldosteronism is most often caused by aldosterone-producing adenoma (APA) and bi-lateral adrenal hyperplasia. Most APAs are caused by somatic mutations of various ion channels and pumps, the most common being the inward-rectifying potassium channel KCNJ5. Germ line mutations of KCNJ5 cause familial hyperaldosteronism type 3 (FH3), which is associated with severe hyperaldosteronism and hypertension. We present an unusual case of FH3 in a young woman, first diagnosed with primary aldosteronism at the age of 6 years, with bilateral adrenal hyperplasia, who underwent unilateral adrenalectomy (left adrenal) to alleviate hyperaldosteronism. However, her hyperaldosteronism persisted. At the age of 26 years, tomography of the remaining adrenal revealed two different adrenal tumors, one of which grew substantially in 4 months; therefore, the adrenal gland was removed. A comprehensive histological, immunohistochemical, and molecular evaluation of various sections of the adrenal gland and in situ visualization of aldosterone, using matrix-assisted laser desorption/ionization imaging mass spectrometry, was performed. Aldosterone synthase (CYP11B2) immunoreactivity was observed in the tumors and adrenal gland. The larger tumor also harbored a somatic ß-catenin activating mutation. Aldosterone visualized in situ was only found in the subcapsular regions of the adrenal and not in the tumors. Collectively, this case of FH3 presented unusual tumor development and histological/molecular findings.

Membrane-type frizzled-related protein regulates lipidome and transcription for photoreceptor function.

Molecular decision-makers of photoreceptor (PRC) membrane organization and gene regulation are critical to understanding sight and retinal degenerations that lead to blindness. Using Mfrprd6 mice, which develop PRC degeneration, we uncovered that membrane-type frizzled-related protein (MFRP) participates in docosahexaenoic acid (DHA, 22:6) enrichment in a manner similar to adiponectin receptor 1 (AdipoR1). Untargeted imaging mass spectrometry demonstrates cell-specific reduction of phospholipids containing 22:6 and very long-chain polyunsaturated fatty acids (VLC-PUFAs) in Adipor1-/- and Mfrprd6 retinas. Gene expression of pro-inflammatory signaling pathways is increased and gene-encoding proteins for PRC function decrease in both mutants. Thus, we propose that both proteins are necessary for retinal lipidome membrane organization, visual function, and to the understanding of the early pathology of retinal degenerative diseases.

Diagnosis of melanoma by imaging mass spectrometry: Development and validation of a melanoma prediction model.

BACKGROUND: The definitive diagnosis of melanocytic neoplasia using solely histopathologic evaluation can be challenging. Novel techniques that objectively confirm diagnoses are needed. This study details the development and validation of a melanoma prediction model from spatially resolved multivariate protein expression profiles generated by imaging mass spectrometry (IMS). METHODS: Three board-certified dermatopathologists blindly evaluated 333 samples. Samples with triply concordant diagnoses were included in this study, divided into a training set (n = 241) and a test set (n = 92). Both the training and test sets included various representative subclasses of unambiguous nevi and melanomas. A prediction model was developed from the training set using a linear support vector machine classification model. RESULTS: We validated the prediction model on the independent test set of 92 specimens (75 classified correctly, 2 misclassified, and 15 indeterminate). IMS detects melanoma with a sensitivity of 97.6% and a specificity of 96.4% when evaluating each unique spot. IMS predicts melanoma at the sample level with a sensitivity of 97.3% and a specificity of 97.5%. Indeterminate results were excluded from sensitivity and specificity calculations. CONCLUSION: This study provides evidence that IMS-based proteomics results are highly concordant to diagnostic results obtained by careful histopathologic evaluation from a panel of expert dermatopathologists.

Visualization of the tissue distribution of fullerenols in zebrafish (Danio rerio) using imaging mass spectrometry.

With the wide application of fullerenols in biomedicine, their environmental exposure risks and toxicity to organisms have been extensively studied. However, there is still a lack of knowledge about the distribution of fullerenols in organisms as an important aspect of their mechanism of toxicity. High-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is an emerging technology for researching the distribution of molecules in biological tissue samples. Using this high-resolution technique, we map the distribution of fullerenols in zebrafish tissues, and the results suggest that fullerenols enter the gill, intestine, and muscle tissues and even permeate the blood-brain barrier, reaching the brain of zebrafish after aquatic exposure. Moreover, from the MS images of fullerenols, the distribution amount of fullerenols is highest in the gill, followed by that in the intestine and the small amount in muscle and brain tissues. As an emerging environmental pollutant, the establishment of this research method will provide a new method for the study of the environmental toxicity of carbon nanomaterials. Our results also indicated that this high-resolution imaging method could be applied to explore the mechanism of interaction between carbon nanomaterials and biological systems at the cellular level in the future.

Distribution of Small Molecular Weight Drugs into the Porcine Lens: Studies on Imaging Mass Spectrometry, Partition Coefficients, and Implications in Ocular Pharmacokinetics.

Lens is the avascular tissue in the eye between the aqueous humor and vitreous. Drug binding to the lens might affect ocular pharmacokinetics, and the binding may also have a pharmacological role in drug-induced cataract and cataract treatment. Drug distribution in the lens has been studied in vitro with many compounds; however, the experimental methods vary, no detailed information on distribution between the lens sublayers exist, and the partition coefficients are reported rarely. Therefore, our objectives were to clarify drug localization in the lens layers and establish partition coefficients for a wide range of molecules. Furthermore, we aimed to illustrate the effect of lenticular drug binding on overall ocular drug pharmacokinetics. We studied the distribution of 16 drugs and three fluorescent dyes in whole porcine lenses in vitro with imaging mass spectrometry and fluorescence microscopy techniques. Furthermore, we determined lens/buffer partition coefficients with the same experimental setup for 28 drugs with mass spectrometry. Finally, the effect of lenticular binding of drugs on aqueous humor drug exposure was explored with pharmacokinetic simulations. After 4 h, the drugs and the dyes distributed only to the outermost lens layers (capsule and cortex). The lens/buffer partition coefficients for the drugs were low, ranging from 0.05 to 0.8. On the basis of the pharmacokinetic simulations, a high lens-aqueous humor partition coefficient increases drug exposure in the lens but does not significantly alter the pharmacokinetics in the aqueous humor. To conclude, the lens seems to act mainly as a physical barrier for drug distribution in the eye, and drug binding to the lens affects mainly the drug pharmacokinetics in the lens.

Analysis of the Heterogeneous Distribution of Amiloride and Propranolol in Dried Blood Spot by UHPLC-FLD and MALDI-IMS.

Dried blood spot (DBS) has lately experienced an increase in its use in bioanalysis due to its several advantages compared with traditional blood sampling methods. Nevertheless, the use of DBS with quantitative purposes is hindered by the heterogeneous distribution of some compounds in the supporting matrix and the dependence of the response on different factors, such as the hematocrit, blood volume, and sampling position. In this study the effect of those factors in the analytical response was investigated by ultra high performance liquid chromatography coupled to fluorescence detection, using amiloride and propranolol as model compounds. The results showed a heterogeneous and drug-dependent distribution of the compounds in the blood spot. While amiloride concentration was higher in the center, propranolol concentration was higher in the periphery of the spot. Besides, the influence of the hematocrit on the quantitative results was observed. MALDI mass spectrometry imaging (MALDI-IMS) has allowed study of the distribution of the two cardiovascular drugs when they were placed in the DBS card using water:methanol solutions, demonstrating that they followed a similar distribution pattern as in blood. This work has showed the potentiality of the MALDI-IMS technique to predict the distribution of the drugs in the DBS card.

Clinical applications of MALDI imaging technologies in cancer and neurodegenerative diseases.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) imaging mass spectrometry (IMS) enables localization of analytes of interest along with histology. More specifically, MALDI-IMS identifies the distributions of proteins, peptides, small molecules, lipids, and drugs and their metabolites in tissues, with high spatial resolution. This unique capacity to directly analyze tissue samples without the need for lengthy sample preparation reduces technical variability and renders MALDI-IMS ideal for the identification of potential diagnostic and prognostic biomarkers and disease gradation. MALDI-IMS has evolved rapidly over the last decade and has been successfully used in both medical and basic research by scientists worldwide. In this review, we explore the clinical applications of MALDI-IMS, focusing on the major cancer types and neurodegenerative diseases. In particular, we re-emphasize the diagnostic potential of IMS and the challenges that must be confronted when conducting MALDI-IMS in clinical settings. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

Imaging mass spectrometry in drug development and toxicology.

During the last decades, imaging mass spectrometry has gained significant relevance in biomedical research. Recent advances in imaging mass spectrometry have paved the way for in situ studies on drug development, metabolism and toxicology. In contrast to whole-body autoradiography that images the localization of radiolabeled compounds, imaging mass spectrometry provides the possibility to simultaneously determine the discrete tissue distribution of the parent compound and its metabolites. In addition, imaging mass spectrometry features high molecular specificity and allows comprehensive, multiplexed detection and localization of hundreds of proteins, peptides and lipids directly in tissues. Toxicologists traditionally screen for adverse findings by histopathological examination. However, studies of the molecular and cellular processes underpinning toxicological and pathologic findings induced by candidate drugs or toxins are important to reach a mechanistic understanding and an effective risk assessment strategy. One of IMS strengths is the ability to directly overlay the molecular information from the mass spectrometric analysis with the tissue section and allow correlative comparisons of molecular and histologic information. Imaging mass spectrometry could therefore be a powerful tool for omics profiling of pharmacological/toxicological effects of drug candidates and toxicants in discrete tissue regions. The aim of the present review is to provide an overview of imaging mass spectrometry, with particular focus on MALDI imaging mass spectrometry, and its use in drug development and toxicology in general.

Early lipid changes in acute kidney injury using SWATH lipidomics coupled with MALDI tissue imaging.

Acute kidney injury (AKI) is one of the leading causes of in-hospital morbidity and mortality, particularly in critically ill patients. Although our understanding of AKI at the molecular level remains limited due to its complex pathophysiology, recent advances in both quantitative and spatial mass spectrometric approaches offer new opportunities to assess the significance of renal metabolomic changes in AKI models. In this study, we evaluated lipid changes in early ischemia-reperfusion (IR)-related AKI in mice by using sequential window acquisition of all theoretical spectra (SWATH)-mass spectrometry (MS) lipidomics. We found a significant increase in two abundant ether-linked phospholipids following IR at 6 h postinjury, a plasmanyl choline, phosphatidylcholine (PC) O-38:1 (O-18:0, 20:1), and a plasmalogen, phosphatidylethanolamine (PE) O-42:3 (O-20:1, 22:2). Both of these lipids correlated with the severity of AKI as measured by plasma creatinine. In addition to many more renal lipid changes associated with more severe AKI, PC O-38:1 elevations were maintained at 24 h post-IR, while renal PE O-42:3 levels decreased, as were all ether PEs detected by SWATH-MS at this later time point. To further assess the significance of this early increase in PC O-38:1, we used matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) to determine that it occurred in proximal tubules, a region of the kidney that is most prone to IR injury and also rich in the rate-limiting enzymes involved in ether-linked phospholipid biosynthesis. Use of SWATH-MS lipidomics in conjunction with MALDI-IMS for lipid localization will help in elucidating the role of lipids in the pathobiology of AKI.

MALDI imaging mass spectrometry as a novel tool for detecting histone modifications in clinical tissue samples.

Histone post-translational modifications (PTMs), histone variants and enzymes responsible for the incorporation or the removal of the PTMs are being increasingly associated with human disease. Combinations of histone PTMs and the specific incorporation of variants contribute to the establishment of cellular identity and hence are potential markers that could be exploited in disease diagnostics and prognostics and therapy response prediction. Due to the scarcity of suitable antibodies and the pre-requirement of tissue homogenization for more advanced analytical techniques, comprehensive information regarding the spatial distribution of these factors at the tissue level has been lacking. MALDI imaging mass spectrometry provides an ideal platform to measure histone PTMs and variants from tissues while maintaining the information about their spatial distribution. Discussed in this review are the relevance of histones in the context of human disease and the contribution of MALDI imaging mass spectrometry in measuring histones in situ.

ESI-MS/MS and MALDI-IMS Localization Reveal Alterations in Phosphatidic Acid, Diacylglycerol, and DHA in Glioma Stem Cell Xenografts.

Glioblastoma (GBM) is the most common adult primary brain tumor. Despite aggressive multimodal therapy, the survival of patients with GBM remains dismal. However, recent evidence has demonstrated the promise of bone marrow-derived mesenchymal stem cells (BM-hMSCs) as a therapeutic delivery vehicle for anti-glioma agents due to their ability to migrate or home to human gliomas. While several studies have demonstrated the feasibility of harnessing the homing capacity of BM-hMSCs for targeted delivery of cancer therapeutics, it is now also evident, based on clinically relevant glioma stem cell (GSC) models of GBMs, that BM-hMSCs demonstrate variable tropism toward these tumors. In this study, we compared the lipid environment of GSC xenografts that attract BM-hMSCs (N = 9) with those that do not attract (N = 9) to identify lipid modalities that are conducive to homing of BM-hMSC to GBMs. We identified lipids directly from tissue by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS) of lipid extracts. Several species of signaling lipids, including phosphatidic acid (PA 36:2, PA 40:5, PA 42:5, and PA 42:7) and diacylglycerol (DAG 34:0, DAG 34:1, DAG 36:1, DAG 38:4, DAG 38:6, and DAG 40:6), were lower in attracting xenografts. Molecular lipid images showed that PA (36:2), DAG (40:6), and docosahexaenoic acid (DHA) were decreased within tumor regions of attracting xenografts. Our results provide the first evidence for lipid signaling pathways and lipid-mediated tumor inflammatory responses in the homing of BM-hMSCs to GSC xenografts. Our studies provide new fundamental knowledge on the molecular correlates of the differential homing capacity of BM-hMSCs toward GSC xenografts.

Direct localisation of molecules in tissue sections of growing antler tips using MALDI imaging.

The astonishing growth rate of deer antlers offers a valuable model for the discovery of novel factors and regulatory systems controlling rapid tissue growth. Numerous molecules have been identified in growing antlers using a variety of techniques. However, little is known about the spatial distribution of these molecules in situ. A technique that has the potential to help in this regard is direct proteomic analysis of tissue sections by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). The present study applied this technique to spatially map molecules in antler tissue sections. Two protonated molecular ions were selected: m/z 6679 and m/z 6200 corresponding to VEGF and thymosin beta-10, respectively. Superimposition of the respective ion images on to histologically stained samples showed distinct spatial distribution across the antler tissue sections which were consistent with the previous reports using in situ hybridization. Two other molecular ions specifically m/z 8100 and m/z 11,800 were also selected, corresponding to reported masses of urocortin precursor and thioredoxin, respectively. As the spatial distribution of these proteins is not specifically known, MALDI-IMS was used as a potential technique to obtain information on their distribution on antler tips. The presence of all these molecules in deer antlers were further confirmed using LC-MS/MS data. The present study also demonstrated that MALDI-IMS could be further used to image antler sections with an extended ion mass range of up to m/z 45,000, thus potentially increasing the ability to discover the distribution of a larger set of molecules that may play an important role in antler growth. We have thus demonstrated that MALDI-IMS is a promising technique for generating molecular maps with high spatial resolution which can aid in evaluating the function of novel molecules during antler growth.

Proteomics for the diagnosis of thyroid lesions: preliminary report.

OBJECTIVE: Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a unique proteomic technology that explores the spatial distribution of biomolecules directly in situ, thus integrating molecular and morphological information. The possibility of correlating distribution maps of multiple analyses with cytological features makes it an ideal research tool for discovering new diagnostic markers. A previous study showed that MALDI-IMS could help discrimination between different types of thyroid lesions, especially papillary thyroid carcinoma (PTC); the present feasibility study on ex vivo fine needle aspiration (FNA) smears describes its potential in detecting new proteomic targets of other thyroid lesions (follicular lesions, medullary carcinoma). METHODS: MALDI-IMS was conducted on ex vivo FNAs obtained from surgical specimens and corresponding in vivo samples. Differences between proteomic profiles of different thyroid lesions were compared. RESULTS: Comparing the protein profiles of hyperplastic nodules obtained from three different patients with each other, and with a new PTC, showed a high degree of concordance, indicating good reproducibility of the IMS technology on cytological samples, suggesting its potential as a tool for biomarker discovery. Furthermore, comparison of the average proteomic profiles of hyperplastic nodules with a Hürthle cell adenoma revealed significant differences, underlying the capability of MALDI-IMS to distinguish between different thyroid lesions. Finally, the proteomic profile of medullary thyroid carcinoma was also characterized. CONCLUSIONS: Our results confirmed the possible role of MALDI-IMS in the search for diagnostic targets of PTC and follicular lesions, which could be applied in larger trials aimed at the identification of proteins, convertible to cost-effective diagnostic tools such as immunohistochemistry. These tests could be used to analyse in vivo cytological smears, improving the preoperative diagnosis of indeterminate thyroid nodules.

On-Tissue Spatial Proteomics Integrating MALDI-MS Imaging with Shotgun Proteomics Reveals Soy Consumption-Induced Protein Changes in a Fragile X Syndrome Mouse Model.

Fragile X syndrome (FXS), the leading cause of inherited intellectual disability and autism, is caused by the transcriptional silencing of the FMR1 gene, which encodes the fragile X messenger ribonucleoprotein (FMRP). FMRP interacts with numerous brain mRNAs that are involved in synaptic plasticity and implicated in autism spectrum disorders. Our published studies indicate that single-source, soy-based diets are associated with increased seizures and autism. Thus, there is an acute need for an unbiased protein marker identification in FXS in response to soy consumption. Herein, we present a spatial proteomics approach integrating mass spectrometry imaging with label-free proteomics in the FXS mouse model to map the spatial distribution and quantify levels of proteins in the hippocampus and hypothalamus brain regions. In total, 1250 unique peptides were spatially resolved, demonstrating the diverse array of peptidomes present in the tissue slices and the broad coverage of the strategy. A group of proteins that are known to be involved in glycolysis, synaptic transmission, and coexpression network analysis suggest a significant association between soy proteins and metabolic and synaptic processes in the Fmr1KO brain. Ultimately, this spatial proteomics work represents a crucial step toward identifying potential candidate protein markers and novel therapeutic targets for FXS.