Effects of Sex and Western Diet on Spatial Lipidomic Profiles for the Hippocampus, Cortex, and Corpus Callosum in Mice Using MALDI MSI.

Diet is inextricably linked to human health and biological functionality. Reduced cognitive function among other health issues has been correlated with a western diet (WD) in mouse models, indicating that increases in neurodegeneration could be fueled in part by a poor diet. In this study, we use matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to spatially map the lipidomic profiles of male and female mice that were fed a high-fat, high-sucrose WD for a period of 7 weeks. Our findings concluded that the cortex and corpus callosum showed significant lipid variation by WD in female mice, while there was little to no variation in the hippocampus, regardless of sex. On the other hand, lipid profiles were significantly affected by sex in all regions. Overall, 83 lipids were putatively identified in the mouse brain; among them, HexCer(40:1;O3) and PE(34:0) were found to have the largest statistical difference based on diet for female mice in the cortex and corpus callosum, respectively. Additional lipid changes are noted and can serve as a metric for understanding the brain's metabolomic response to changes in diet, particularly as it relates to disease.

Optimized combination of MALDI MSI and immunofluorescence for neuroimaging of lipids within cellular microenvironments.

Proper neurological function relies on the cellular and molecular microenvironment of the brain, with perturbations of this environment leading to neurological disorders. However, studying the microenvironments of neurological tissue has proven difficult because of its inherent complexity. Both the cell type and metabolomic underpinnings of the cell have crucial functional roles, thus making multimodal characterization methods key to acquiring a holistic view of the brain's microenvironment. This study investigates methods for combining matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and immunofluorescence (IF) microscopy to enable the concurrent investigation of cell types and lipid profiles on the same sample. In brief, 1,5-diaminonaphthalene (DAN), α-cyano-4-hydroxy-cinnamic acid (CHCA), and 2,5-dihydroxybenzoic acid (DHB) were tested in addition to instrument-specific parameters for compatibility with IF. Alternatively, the effects of IF protocols on MALDI MSI were also tested, showing significant signal loss with all tested permutations. Ultimately, the use of CHCA for MALDI MSI resulted in the best IF images, while the use of DAN gave the lowest quality IF images. Overall, increasing the laser power and number of shots per laser burst resulted in the most tissue ablation. However, optimized parameter settings allowed for minimal tissue ablation while maintaining sufficient MALDI MSI signal.

Sample Preparation Method for MALDI Mass Spectrometry Imaging of Fresh-Frozen Spines.

Technologies assessing the lipidomics, genomics, epigenomics, transcriptomics, and proteomics of tissue samples at single-cell resolution have deepened our understanding of physiology and pathophysiology at an unprecedented level of detail. However, the study of single-cell spatial metabolomics in undecalcified bones faces several significant challenges, such as the fragility of bone, which often requires decalcification or fixation leading to the degradation or removal of lipids and other molecules. As such, we describe a method for performing mass spectrometry imaging on undecalcified spine that is compatible with other spatial omics measurements. In brief, we use fresh-frozen rat spines and a system of carboxyl methylcellulose embedding, cryofilm, and polytetrafluoroethylene rollers to maintain tissue integrity while avoiding signal loss from variations in laser focus and artifacts from traditional tissue processing. This reveals various tissue types and lipidomic profiles of spinal regions at 10 µm spatial resolutions using matrix-assisted laser desorption/ionization mass spectrometry imaging. We expect this method to be adapted and applied to the analysis of the spinal cord, shedding light on the mechanistic aspects of cellular heterogeneity, development, and disease pathogenesis underlying different bone-related conditions and diseases. This study furthers the methodology for high spatial metabolomics of spines and adds to the collective efforts to achieve a holistic understanding of diseases via single-cell spatial multiomics.

Spatial lipidomics of fresh-frozen spines.

Technologies assessing the lipidomics, genomics, epigenomics, transcriptomics, and proteomics of tissue samples at single-cell resolution have deepened our understanding of physiology and pathophysiology at an unprecedented level of detail. However, the study of single-cell spatial metabolomics in undecalcified bones faces several significant challenges, such as the fragility of bone which often requires decalcification or fixation leading to the degradation or removal of lipids and other molecules and. As such, we describe a method for performing mass spectrometry imaging on undecalcified spine that is compatible with other spatial omics measurements. In brief, we use fresh-freeze rat spines and a system of carboxyl methylcellulose embedding, cryofilm, and polytetrafluoroethylene rollers to maintain tissue integrity, while avoiding signal loss from variations in laser focus and artifacts from traditional tissue processing. This reveals various tissue types and lipidomic profiles of spinal regions at 10 µm spatial resolutions using matrix-assisted laser desorption/ionization mass spectrometry imaging. We expect this method to be adapted and applied to the analysis of spinal cord, shedding light on the mechanistic aspects of cellular heterogeneity, development, and disease pathogenesis underlying different bone-related conditions and diseases. This study furthers the methodology for high spatial metabolomics of spines, as well as adds to the collective efforts to achieve a holistic understanding of diseases via single-cell spatial multi-omics.

Organ Mapping Antibody Panels: a community resource for standardized multiplexed tissue imaging.

Multiplexed antibody-based imaging enables the detailed characterization of molecular and cellular organization in tissues. Advances in the field now allow high-parameter data collection (>60 targets); however, considerable expertise and capital are needed to construct the antibody panels employed by these methods. Organ mapping antibody panels are community-validated resources that save time and money, increase reproducibility, accelerate discovery and support the construction of a Human Reference Atlas.

MALDI IMS-Derived Molecular Contour Maps: Augmenting Histology Whole-Slide Images.

Imaging mass spectrometry (IMS) provides untargeted, highly multiplexed maps of molecular distributions in tissue. Ion images are routinely presented as heatmaps and can be overlaid onto complementary microscopy images that provide greater context. However, heatmaps use transparency blending to visualize both images, obscuring subtle quantitative differences and distribution gradients. Here, we developed a contour mapping approach that combines information from IMS ion intensity distributions with that of stained microscopy. As a case study, we applied this approach to imaging data from Staphylococcus aureus-infected murine kidney. In a univariate, or single molecular species, use-case of the contour map representation of IMS data, certain lipids colocalizing with regions of infection were selected using Pearson's correlation coefficient. Contour maps of these lipids overlaid with stained microscopy showed enhanced visualization of lipid distributions and spatial gradients in and around the bacterial abscess as compared to traditional heatmaps. The full IMS data set comprising hundreds of individual ion images was then grouped into a smaller subset of representative patterns using non-negative matrix factorization (NMF). Contour maps of these multivariate NMF images revealed distinct molecular profiles of the major abscesses and surrounding immune response. This contour mapping workflow also enabled a molecular visualization of the transition zone at the host-pathogen interface, providing potential clues about the spatial molecular dynamics beyond what histological staining alone provides. In summary, we developed a new IMS-based contour mapping approach to augment classical stained microscopy images, providing an enhanced and more interpretable visualization of IMS-microscopy multimodal molecular imaging data sets.

Rapid Multivariate Analysis Approach to Explore Differential Spatial Protein Profiles in Tissue.

Spatially targeted proteomics analyzes the proteome of specific cell types and functional regions within tissue. While spatial context is often essential to understanding biological processes, interpreting sub-region-specific protein profiles can pose a challenge due to the high-dimensional nature of the data. Here, we develop a multivariate approach for rapid exploration of differential protein profiles acquired from distinct tissue regions and apply it to analyze a published spatially targeted proteomics data set collected from Staphylococcus aureus-infected murine kidney, 4 and 10 days postinfection. The data analysis process rapidly filters high-dimensional proteomic data to reveal relevant differentiating species among hundreds to thousands of measured molecules. We employ principal component analysis (PCA) for dimensionality reduction of protein profiles measured by microliquid extraction surface analysis mass spectrometry. Subsequently, k-means clustering of the PCA-processed data groups samples by chemical similarity. Cluster center interpretation revealed a subset of proteins that differentiate between spatial regions of infection over two time points. These proteins appear involved in tricarboxylic acid metabolomic pathways, calcium-dependent processes, and cytoskeletal organization. Gene ontology analysis further uncovered relationships to tissue damage/repair and calcium-related defense mechanisms. Applying our analysis in infectious disease highlighted differential proteomic changes across abscess regions over time, reflecting the dynamic nature of host-pathogen interactions.

Imaging mass spectrometry reveals complex lipid distributions across Staphylococcus aureus biofilm layers.

Introduction: Although Staphylococcus aureus is the leading cause of biofilm-related infections, the lipidomic distributions within these biofilms is poorly understood. Here, lipidomic mapping of S. aureus biofilm cross-sections was performed to investigate heterogeneity between horizontal biofilm layers. Methods: S. aureus biofilms were grown statically, embedded in a mixture of carboxymethylcellulose/gelatin, and prepared for downstream matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Trapped ion mobility spectrometry (TIMS) was also applied prior to mass analysis. Results: Implementation of TIMS led to a âˆ¼ threefold increase in the number of lipid species detected. Washing biofilm samples with ammonium formate (150 mM) increased signal intensity for some bacterial lipids by as much as tenfold, with minimal disruption of the biofilm structure. MALDI TIMS IMS revealed that most lipids localize primarily to a single biofilm layer, and species from the same lipid class such as cardiolipins CL(57:0) - CL(66:0) display starkly different localizations, exhibiting between 1.5 and 6.3-fold intensity differences between layers (n = 3, p < 0.03). No horizontal layers were observed within biofilms grown anaerobically, and lipids were distributed homogenously. Conclusions: High spatial resolution analysis of S. aureus biofilm cross-sections by MALDI TIMS IMS revealed stark lipidomic heterogeneity between horizontal S. aureus biofilm layers demonstrating that each layer was molecularly distinct. Finally, this workflow uncovered an absence of layers in biofilms grown under anaerobic conditions, possibly indicating that oxygen contributes to the observed heterogeneity under aerobic conditions. Future applications of this workflow to study spatially localized molecular responses to antimicrobials could provide new therapeutic strategies.

Strain-Release Pentafluorosulfanylation and Tetrafluoro(aryl)sulfanylation of [1.1.1]Propellane: Reactivity and Structural Insight.

We leveraged the recent increase in synthetic accessibility of SF5 Cl and Ar-SF4 Cl compounds to combine chemistry of the SF5 and SF4 Ar groups with strain-release functionalization. By effectively adding SF5 and SF4 Ar radicals across [1.1.1]propellane, we accessed structurally unique bicyclopentanes, bearing two distinct elements of bioisosterism. Upon evaluating these "hybrid isostere" motifs in the solid state, we measured exceptionally short transannular distances; in one case, the distance rivals the shortest nonbonding C⋅⋅⋅C contact reported to date. This prompted SC-XRD and DFT analyses that support the notion that a donor-acceptor interaction involving the "wing" C-C bonds is playing an important role in stabilization. Thus, these heretofore unknown structures expand the palette for highly coveted three-dimensional fluorinated building blocks and provide insight to a more general effect observed in bicyclopentanes.

Highly multiplexed, label-free proteoform imaging of tissues by individual ion mass spectrometry.

Imaging of proteoforms in human tissues is hindered by low molecular specificity and limited proteome coverage. Here, we introduce proteoform imaging mass spectrometry (PiMS), which increases the size limit for proteoform detection and identification by fourfold compared to reported methods and reveals tissue localization of proteoforms at <80-µm spatial resolution. PiMS advances proteoform imaging by combining ambient nanospray desorption electrospray ionization with ion detection using individual ion mass spectrometry. We demonstrate highly multiplexed proteoform imaging of human kidney, annotating 169 of 400 proteoforms of <70 kDa using top-down MS and a database lookup of ~1000 kidney candidate proteoforms, including dozens of key enzymes in primary metabolism. PiMS images reveal distinct spatial localizations of proteoforms to both anatomical structures and cellular neighborhoods in the vasculature, medulla, and cortex regions of the human kidney. The benefits of PiMS are poised to increase proteome coverage for label-free protein imaging of tissues.

Referenced Kendrick Mass Defect Annotation and Class-Based Filtering of Imaging MS Lipidomics Experiments.

Because of their diverse functionalities in cells, lipids are of primary importance when characterizing molecular profiles of physiological and disease states. Imaging mass spectrometry (IMS) provides the spatial distributions of lipid populations in tissues. Referenced Kendrick mass defect (RKMD) analysis is an effective mass spectrometry (MS) data analysis tool for classification and annotation of lipids. Herein, we extend the capabilities of RKMD analysis and demonstrate an integrated method for lipid annotation and chemical structure-based filtering for IMS datasets. Annotation of lipid features with lipid molecular class, radyl carbon chain length, and degree of unsaturation allows image reconstruction and visualization based on each structural characteristic. We show a proof-of-concept application of the method to a computationally generated IMS dataset and validate that the RKMD method is highly specific for lipid components in the presence of confounding background ions. Moreover, we demonstrate an application of the RKMD-based annotation and filtering to matrix-assisted laser desorption/ionization (MALDI) IMS lipidomic data from human kidney tissue analysis.

High Spatial Resolution MALDI Imaging Mass Spectrometry of Fresh-Frozen Bone.

Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to molecular alterations giving rise to debilitating diseases like rheumatoid arthritis and osteomyelitis. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) facilitate the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of osseous tissue for MALDI IMS has been difficult due to its mineralized composition and heterogeneous morphology, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs, allowing the distribution of endogenous lipids to be linked to tissue structures and cell types. Adhesive-bound bone sections were mounted onto conductive glass slides with microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. High spatial resolution (10 µm) MALDI IMS was employed to characterize lipid distributions, and use of complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to the bone marrow, adipose tissue, marrow adipose tissue, and muscle. Further, sphingomyelin(42:1) was abundant in megakaryocytes, whereas sphingomyelin(42:2) was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage with spatial relevance in a preserved native bone microenvironment.

Highly multiplexed immunofluorescence of the human kidney using co-detection by indexing.

The human kidney is composed of many cell types that vary in their abundance and distribution from normal to diseased organ. As these cell types perform unique and essential functions, it is important to confidently label each within a single tissue to accurately assess tissue architecture and microenvironments. Towards this goal, we demonstrate the use of co-detection by indexing (CODEX) multiplexed immunofluorescence for visualizing 23 antigens within the human kidney. Using CODEX, many of the major cell types and substructures, such as collecting ducts, glomeruli, and thick ascending limb, were visualized within a single tissue section. Of these antibodies, 19 were conjugated in-house, demonstrating the flexibility and utility of this approach for studying the human kidney using custom and commercially available antibodies. We performed a pilot study that compared both fresh frozen and formalin-fixed paraffin-embedded healthy non-neoplastic and diabetic nephropathy kidney tissues. The largest cellular differences between the two groups was observed in cells labeled with aquaporin 1, cytokeratin 7, and α-smooth muscle actin. Thus, our data show the power of CODEX multiplexed immunofluorescence for surveying the cellular diversity of the human kidney and the potential for applications within pathology, histology, and building anatomical atlases.

Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging.

Tissues and organs are composed of distinct cell types that must operate in concert to perform physiological functions. Efforts to create high-dimensional biomarker catalogs of these cells have been largely based on single-cell sequencing approaches, which lack the spatial context required to understand critical cellular communication and correlated structural organization. To probe in situ biology with sufficient depth, several multiplexed protein imaging methods have been recently developed. Though these technologies differ in strategy and mode of immunolabeling and detection tags, they commonly utilize antibodies directed against protein biomarkers to provide detailed spatial and functional maps of complex tissues. As these promising antibody-based multiplexing approaches become more widely adopted, new frameworks and considerations are critical for training future users, generating molecular tools, validating antibody panels, and harmonizing datasets. In this Perspective, we provide essential resources, key considerations for obtaining robust and reproducible imaging data, and specialized knowledge from domain experts and technology developers.

Protocol for multimodal analysis of human kidney tissue by imaging mass spectrometry and CODEX multiplexed immunofluorescence.

Here, we describe the preservation and preparation of human kidney tissue for interrogation by histopathology, imaging mass spectrometry, and multiplexed immunofluorescence. Custom image registration and integration techniques are used to create cellular and molecular atlases of this organ system. Through careful optimization, we ensure high-quality and reproducible datasets suitable for cross-patient comparisons that are essential to understanding human health and disease. Moreover, each of these steps can be adapted to other organ systems or diseases, enabling additional atlas efforts.

Identification of Lipid Heterogeneity and Diversity in the Developing Human Brain.

The lipidome is currently understudied but fundamental to life. Within the brain, little is known about cell-type lipid heterogeneity, and even less is known about cell-to-cell lipid diversity because it is difficult to study the lipids within individual cells. Here, we used single-cell mass spectrometry-based protocols to profile the lipidomes of 154 910 single cells across ten individuals consisting of five developmental ages and five brain regions, resulting in a unique lipid atlas available via a web browser of the developing human brain. From these data, we identify differentially expressed lipids across brain structures, cortical areas, and developmental ages. We inferred lipid profiles of several major cell types from this data set and additionally detected putative cell-type specific lipids. This data set will enable further interrogation of the developing human brain lipidome.

Integrating ion mobility and imaging mass spectrometry for comprehensive analysis of biological tissues: A brief review and perspective.

Imaging mass spectrometry (IMS) technologies are capable of mapping a wide array of biomolecules in diverse cellular and tissue environments. IMS has emerged as an essential tool for providing spatially targeted molecular information due to its high sensitivity, wide molecular coverage, and chemical specificity. One of the major challenges for mapping the complex cellular milieu is the presence of many isomers and isobars in these samples. This challenge is traditionally addressed using orthogonal liquid chromatography (LC)-based analysis, though, common approaches such as chromatography and electrophoresis are not able to be performed at timescales that are compatible with most imaging applications. Ion mobility offers rapid, gas-phase separations that are readily integrated with IMS workflows in order to provide additional data dimensionality that can improve signal-to-noise, dynamic range, and specificity. Here, we highlight recent examples of ion mobility coupled to IMS and highlight their importance to the field.

Multimodal Imaging Mass Spectrometry: Next Generation Molecular Mapping in Biology and Medicine.

Imaging mass spectrometry has become a mature molecular mapping technology that is used for molecular discovery in many medical and biological systems. While powerful by itself, imaging mass spectrometry can be complemented by the addition of other orthogonal, chemically informative imaging technologies to maximize the information gained from a single experiment and enable deeper understanding of biological processes. Within this review, we describe MALDI, SIMS, and DESI imaging mass spectrometric technologies and how these have been integrated with other analytical modalities such as microscopy, transcriptomics, spectroscopy, and electrochemistry in a field termed multimodal imaging. We explore the future of this field and discuss forthcoming developments that will bring new insights to help unravel the molecular complexities of biological systems, from single cells to functional tissue structures and organs.

Resolving the Complexity of Spatial Lipidomics Using MALDI TIMS Imaging Mass Spectrometry.

Lipids are a structurally diverse class of molecules with important biological functions including cellular signaling and energy storage. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows for direct mapping of biomolecules in tissues. Fully characterizing the structural diversity of lipids remains a challenge due to the presence of isobaric and isomeric species, which greatly complicates data interpretation when only m/z information is available. Integrating ion mobility separations aids in deconvoluting these complex mixtures and addressing the challenges of lipid IMS. Here, we demonstrate that a MALDI quadrupole time-of-flight (Q-TOF) mass spectrometer with trapped ion mobility spectrometry (TIMS) enables a >250% increase in the peak capacity during IMS experiments. MALDI TIMS-MS separation of lipid isomer standards, including sn backbone isomers, acyl chain isomers, and double-bond position and stereoisomers, is demonstrated. As a proof of concept, in situ separation and imaging of lipid isomers with distinct spatial distributions were performed using tissue sections from a whole-body mouse pup.

Spatial Metabolomics of the Human Kidney using MALDI Trapped Ion Mobility Imaging Mass Spectrometry.

Low molecular weight metabolites are essential for defining the molecular phenotypes of cells. However, spatial metabolomics tools often lack the sensitivity, specify, and spatial resolution to provide comprehensive descriptions of these species in tissue. MALDI imaging mass spectrometry (IMS) of low molecular weight ions is particularly challenging as MALDI matrix clusters are often nominally isobaric with multiple metabolite ions, requiring high resolving power instrumentation or derivatization to circumvent this issue. An alternative to this is to perform ion mobility separation before ion detection, enabling the visualization of metabolites without the interference of matrix ions. Additional difficulties surrounding low weight metabolite visualization include high resolution imaging, while maintaining sufficient ion numbers for broad and representative analysis of the tissue chemical complement. Here, we use MALDI timsTOF IMS to image low molecular weight metabolites at higher spatial resolution than most metabolite MALDI IMS experiments (20 µm) while maintaining broad coverage within the human kidney. We demonstrate that trapped ion mobility spectrometry (TIMS) can resolve matrix peaks from metabolite signal and separate both isobaric and isomeric metabolites with different distributions within the kidney. The added ion mobility data dimension dramatically increased the peak capacity for spatial metabolomics experiments. Through this improved sensitivity, we have found >40 low molecular weight metabolites in human kidney tissue, such as argininic acid, acetylcarnitine, and choline that localize to the cortex, medulla, and renal pelvis, respectively. Future work will involve further exploring metabolomic profiles of human kidneys as a function of age, sex, and race.