Mass Spectrometry Imaging Research Articles

Introduction: Arterial calcification contributes significantly to cardiovascular morbidity, yet its metabolic mechanisms remain poorly understood. This study aimed to characterize the spatial metabolic landscape of vascular calcification using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in both human and animal tissues. Methods: Fresh calcified coronary arteries from cadaver hearts (n=8) and femoral arteries from a porcine model of arterial calcification (n=10, including calcified and controls) were snap-frozen and underwent untargeted metabolomic profiling using a UV-laser MALDI source (Spectroglyph LLC) coupled with a Thermo Q Exactive HF-X Orbitrap MS. Adjacent tissue sections were stained with Von Kossa to identify calcified regions (VK+), which were annotated and co-registered with MALDI-MSI datasets to correlate spatial metabolic features with mineralization. Metabolite analysis (>1550 features) was performed using QuPath and SCiLS, with statistical comparison by PCA and Pearson correlation (p<0.001). Results: In human coronary arteries, PCA plot indicated distinct metabolic profile between VK- and VK+ samples (Fig.1A). Spatial metabolomics analysis revealed a distinct set of 79 metabolites adjacent to calcified regions, primarily glycerophospholipids (38), sphingolipids (6), amino acid derivatives (5), fatty acids (3), and nucleosides&nucleotides (2), with 49 significantly elevated and spatially localized to calcified regions. In the porcine model, difference between VK+ and VK- regions was found (Fig.1B) and a robust panel of 90 metabolites was significantly associated with calcification (|r|>0.5), the majority of which were amino acid derivatives (32), fatty acids (6), nucleosides and nucleosides (5), and glycerophospholipids (4), consistent with human coronary artery results. Notably, 22 metabolites, including adenosine monophosphate, pyruvate, and phosphatidylinositol (20:2/16:0), were associated with pathways such as glycolysis and purine metabolism, mirrored the metabolic alterations observed in human tissues, supporting the translational relevance of the model. Conclusion: This study demonstrates the power of MALDI-MSI for high-resolution spatial metabolomic profiling of arterial calcification. The identified metabolite signatures provide new insights into the pathobiology of arterial calcification and may inform future therapeutic strategies targeting vascular mineralization and early biomarker discovery.

Increasing ion abundance in mass spectrometry is essential for enhancing detection, quantification, and understanding of biomolecules involved in key cellular processes. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI), MALDI, and nano-DESI have shown improved ion abundance when integrating specific concentrations of ammonium fluoride into the sample preparation and/or ionization steps. Herein, the importance of electronegativity and atomic size for the proposed mechanism of increased ion abundance is evaluated by testing a variety of ammonium halide salts. Ammonium fluoride was confirmed to result in the largest increase in ion abundance and was subsequently used to perform quantitative mass spectrometry imaging (qMSI) of glutathione (GSH) in healthy mouse liver tissue. Using ~70 µM NH4F as an ESI dopant, up to a ~ two-fold increase in ion abundance was observed for these biomolecules, as well as an improvement in the limit of detection, detection frequency, and quantification of endogenous GSH in tissue.

Background: Cardiomyocyte proliferation, the fundamental mechanism of heart development and regeneration, is active in infants. We investigated cardiomyocyte proliferation in infants with heart disease. Objective/Hypothesis: Infants with tetralogy of Fallot (ToF, the most common type of cyanotic congenital heart disease) or heart failure (HF) exhibit altered cardiomyocyte proliferation. Methods: The DNA replication marker, 15 N-thymidine, was administered to infants with unrepaired ToF (n = 13) and to infants with heart failure waiting for transplantation (n = 5). One of the infants with ToF was also treated with the beta-blocker, propranolol. Myocardial samples were ascertained at the time of ToF surgery, placement or removal of left ventricular assist devices (LVAD), and heart transplantation. Retention of 15 N-thymidine in cells that had undergone DNA replication was assessed using Multi-Isotope Imaging Mass Spectrometry (MIMS). Polyploidy was quantified to identify post-mitotic, non-dividing cardiomyocytes. Results: Within the first 4 months after birth, 0.74 billion new cardiomyocytes were generated—3.4-fold higher than after the age of 4 months (P = 0.0417). Average per nucleus DNA content in 15 N-labeled cardiomyocytes in ToF infants (n = 11) and HF infants (n = 3) was significantly greater (1.38-fold) than that in individuals without heart disease (n = 11, P = 0.0358), indicating increased formation of post-mitotic cardiomyocytes in diseased hearts. An infant with HF received 15 N-thymidine while on LVAD. Despite clinical improvement and LVAD removal, cardiomyocyte generation remained below age-appropriate levels. In contrast, a ToF infant treated with propranolol, showed a reduced incidence of polyploid nuclei, suggesting enhanced cardiomyocyte division in response to β-blockade. Conclusion: Infants with heart disease generate new cardiomyocytes, with the majority of this occurring in the first four months after birth, which identifies a window for regenerative therapies. Beta-blockade may promote cardiomyocyte division, highlighting a direction for regenerative therapy.

Exploring the metabolic characteristics of different plant organs and tissues at a spatial level can help us to better understand the functional mechanisms of biological tissues and cells. Mass spectrometry imaging (MSI) provides a reliable tool for this purpose. However, its application for high-resolution metabolic mapping across various plant organs remains a significant challenge due to the intrinsic biological properties of plant samples and unfavorable analysis conditions. This study aimed to develop a novel MSI platform that can expand more diverse plant samples in spatial metabolomics research and enhance the detection efficiency of plant metabolites. The platform (AMG-LDI-MSI) based on an Au nanoparticles-loaded MoS2 and doped graphene oxide (Au@MoS2/GO) flexible film substrate combined with laser desorption/ionization (LDI)-MSI was established to enhance the detection and visualization of metabolites in various plant tissues. It has a non-sectioning, matrix-free, dual-ion mode imaging strategy, enabling high-throughput detection of metabolites and high-resolution molecular imaging within a micrometer scale. The Au@MoS2/GO as a new substrate can offer high sensitivity and molecular coverage for diverse plant metabolites (10 classes) under the positive and negative ion modes. Moreover, the AMG-LDI-MSI platform overcomes the limitations of plant tissues (e.g., fragile leaf, water-rich fruit, or lignified roots) for in situ imaging. We successfully applied the platform to map the metabolite spatial dynamics in different types of fresh tissues (rhizome, main root, branch root, fruit, leaf, and root nodule) from medicinal plants, obtained the high-quality mass spectral imaging data, and demonstrated the universality and applicability of the platform to multiple plant tissues. These results demonstrate the significant advantages of enhancing the detection of multiple tissue metabolites in plants and their high-resolution imaging. It has overcome the limitations of previously reported MSI methods, suggesting that it could become a widely used tool for deciphering metabolic networks in plant biology.

Metabolic heavyweight: thick ascending limb or proximal tubule? Next round.

Understanding kidney metabolic heterogeneity is critical for unraveling diabetic nephropathy (DN) pathogenesis. While Huangqi Guizhi Wuwu decoction (HGD) shows efficacy against DN, its spatial metabolic effects remain unknown. We integrated liquid chromatography-mass spectrometry (LC-MS) metabolomics, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), and tandem mass tag (TMT) proteomics to map systemic and microregional metabolic landscapes in HGD-treated db/db mice. Spatial metabolomics identified seven compartmentalized metabolites: citrulline, dibutyl phthalate, and hydroxykynurenine showed pan-renal distribution, whereas riboflavin, γ-glutamylcysteine, 7-methylguanine, and L-4-hydroxyphenylglycine localized specifically to the cortical outer layer. HGD restored glomerular architecture, reduced hyperglycemia, and normalized glycated hemoglobin, with MALDI-MSI visualizing cortical metabolite redistribution. Proteomics revealed 10 differentially expressed proteins, and multi-omics integration linked HGD's effects to riboflavin metabolism, arginine biosynthesis, and pancreatic secretion pathways. Crucially, spatial correlation analysis uncovered a cortex-specific metabolic axis between riboflavin and citrulline, indicating compartmentalized cross-talk between vitamin metabolism and urea cycle regulation. This establishes HGD as a spatial metabolic modulator rectifying microregional imbalance in DN and demonstrates how MALDI-MSI-guided multi-omics deciphers tissue-specific herbal pharmacodynamics, providing a paradigm for investigating traditional medicine through spatially resolved molecular mapping.

Particle size-dependent neurotoxicity of microplastics in zebrafish (Danio rerio): Spatially resolved lipidomics links metabolic dysregulation to neurological disorders.

Mass spectrometry imaging for in-situ evaluating the lipid metabolic heterogeneity of 5-fluorouracil in HepG2 spheroids

Cardiomyopathy is a common complication of sepsis that contributes to increased morbidity and mortality. However, the molecular mechanisms underlying septic cardiomyopathy are poorly understood. Dichloroacetate (DCA) improves mitochondrial respiration and survival in a mouse model of sepsis by inhibiting pyruvate dehydrogenase kinase which inactivates pyruvate dehydrogenase (PDH) through phosphorylation of its subunits. In this study, we explore the role of DCA in septic cardiac dysfunction using a murine sepsis model. Cecal ligation and puncture (CLP) was performed in mice to investigate molecular and echocardiographic response to sepsis. DCA was administered to test the effects of PDH activation on cardiac performance during early and late sepsis and myocardial metabolic substrate production. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry was used to reveal spatial alterations in metabolism. CLP significantly increased phosphorylation of the PDH E1α subunit (PDH inactivation), and DCA treatment reduced PDH E1α phosphorylation (PDH activation) to baseline without affecting total PDH E1α levels. Administration of DCA at the time of CLP improved cardiac preload and stroke volume without affecting cardiac contractility at 12 h after CLP. However, there was a significant increase in cardiac contractility at 30 h after DCA administration independent of cardiac loading conditions. This improved cardiac function after DCA administration was associated with a trend toward decreased production of metabolic intermediates such as ketogenic amino acids, succinate, and palmitoyl carnitine. Imaging mass spectrometry revealed an increase in itaconate expression upon CLP that was mitigated by DCA administration. Our findings revealed that sepsis decreased PDH activity in cardiac tissue. Rebalancing PDH activity with DCA improved cardiac performance after CLP. While imaging mass spectrometry identified changes in itaconate concentration and enabled detection of tricarboxylic acid cycle metabolites, further investigation is necessary to determine whether DCA is an effective therapeutic agent for septic cardiomyopathy.

Spatiotemporal Proteomics: Unveiling Evolving Molecular Landscapes in Inflammatory Bowel Disease and Associated Colorectal Cancer.

Investigation of metabolism and spatial distribution of metabolites of metalaxyl and spirotetramat after root uptake in maize using HPLC-HRMS and MALDI-MSI.

The distribution of Danqi tongmai tablet in rats at different timeframe by UHPLC-LTQ-orbitrap mass spectrometry and nanospray desorption electrospray ionization mass spectrometry imaging.

Mass spectrometry imaging (MSI) enables the visualization of hundreds to thousands of analytes in biological tissues. MSI is also capable of mapping time-dependent processes that, combined with these static metabolite profiles, provides a clearer picture of the molecular underpinnings of tissue function. This perspective is organized into sections demonstrating how MSI-based methods can provide unique functional data on systems ranging from single step enzyme-catalyzed transformations to complex metabolic network activities and cellular dynamics. This multisystem capability can be exploited to provide detailed descriptions of the molecular mechanisms contributing to tissue function. An aspect missing in many studies are corresponding maps of tissue microenvironments including oxygenation and pH which influence functional activities. Some progress has been made to map hypoxic and acidic tissue using MSI methods, but further development is needed. This includes pairing in vivo MSI functional studies to in vitro models. Additionally, integrating the capabilities of other imaging methods, such as magnetic resonance and vibrational spectroscopy, that are proven to detect tissue microenvironments, with dynamic MSI methods offer a route to match environment with functional activities. The combination of static molecular profiles, metabolic and cellular dynamics, and environmental mapping will provide the most detailed understanding of tissue function.

Pesticides are essential for crop protection but can markedly reshape plant metabolism with implications for food quality and safety. This work introduces an integrated strategy that combines untargeted ultrahigh-performance liquid chromatography-ultrahigh-resolution mass spectrometry (UHPLC-UHRMS) with two- and three-dimensional mass spectrometry imaging using laser ablation remote atmospheric pressure photoionization/chemical ionization (LARAPPI/CI-2D/3D-MSI) to elucidate global and spatial metabolic responses of radish to pesticide exposure. The results reveal compound- and dose-dependent effects: field-relevant concentrations cause minor metabolic perturbations, whereas 100-fold higher doses induce systemic reprogramming of amino acid, carbohydrate, lipid, and secondary metabolism. MSI uncovers distinct tissue- and depth-specific patterns of metabolic alteration, demonstrating nonadditive responses to pesticide mixtures. By linking molecular profiling with spatial metabolite mapping, this work advances the mechanistic understanding of plant stress responses and provides a framework for evaluating the metabolic consequences of pesticide regimes on crop physiology and food safety.

Mass spectrometry imaging (MSI) can be used to survey numerous molecular species from a wide variety of surfaces, including biological tissue sections. Atmospheric-pressure (AP) infrared laser-ablation plasma postionization (IR-PPI) has recently been shown to allow matrix free analysis of small molecules from both fresh frozen and formalin fixed paraffin embedded (FFPE) tissue. Detected ion intensities in IR-PPI as well as other AP inlet modalities such as desorption electrospray ionization (DESI) show a strong dependence on the inlet capillary temperature. In this study, the relationship between detected ion intensity and inlet capillary temperature is evaluated, between room temperature and 650 °C, for analyte pipetted on various substrates, as well as fresh frozen and FFPE tissue, by IR-PPI. Temperature trends for exemplar ions of interest show a variety of dependencies with optimal temperatures observed throughout this temperature range. For example, detection of lactate [M-H]- m/z 89.0244 is optimal at ∼100 °C, glutamine [M-H]- m/z 145.0618 at ∼250 °C, arachidonic acid [M-H]- m/z 303.2324 at ∼150 °C and PI(18:0/20:4) [M-H]- m/z 885.5488 at ∼500 °C. Data reduction and clustering of these data by uniform manifold approximation and projection (UMAP) and k-means provides a summary of all temperature trends within the data and association of different ions with these trends are presented. Finally, the implications of different inlet capillary temperature settings in tissue MSI are demonstrated by comparing detected glucose and lactate ion intensities in response to different inlet temperatures in mouse brain. The choice and control of inlet temperature are shown to be critical variables for the interpretation of biological MSI data in AP modalities.

Mass spectrometry imaging (MSI) enables label-free molecular mapping in tissues but presents challenges for spatial segmentation due to high dimensionality, nonlinear spectral variation, and tissue heterogeneity. Traditional unsupervised clustering methods often rely on predefined cluster numbers and overlook spatial information, yielding fragmented or biologically implausible results. We introduce MSInet, a self-supervised deep learning framework for robust, annotation-free MSI segmentation. MSInet combines two strategies within a convolutional neural network: patch-wise contrastive learning to capture global semantic relationships, and superpixel-guided refinement to enforce local spatial consistency. This dual-consistency design simultaneously enhances global context awareness and local boundary precision during training. MSInet was evaluated on MALDI-MSI of mouse brain, DESI-MSI of renal tumor, and a synthetic data set with ground truth. It consistently outperformed state-of-the-art methods (e.g., t-SNE + k-means, CNNAE + region-growing, and GCN-based models), achieving higher accuracy and biological fidelity. On simulated data, MSInet achieved an Adjusted Rand Index of 0.89 and Normalized Mutual Information of 0.86, with ∼25.8% ARI improvement over baselines. It also precisely delineated complex anatomical subregions in the brain (Silhouette Coefficient = 0.78) and distinguished tumor, necrosis, and healthy regions in renal tissues, closely aligning with histological references. MSInet further demonstrated robustness to MSI noise. By integrating global and local contextual modeling in a self-supervised architecture, MSInet offers a powerful, scalable solution for accurate and biologically meaningful MSI segmentation, with broad potential for spatial omics and biomedical applications.

Investigating the Quantitative Structure-Ionization Efficiency Relationship of Small Molecules and Lipids in the Presence of Ammonium Fluoride in MALDI-TIMS-QTOF Mass Spectrometry Imaging.

Hepatocyte phospholipase D1 ( PLD1) knockout alleviates metabolic dysfunction-associated steatotic liver disease (MASLD) in mice, but the underlying mechanism is largely unknown. In this study, the mice were divided into four groups: Con (wild-type mice with normal control diet), HFHC (wild-type mice with high-fat diet), Con_KO (hepatocyte PLD1-knockout mice with normal control diet), and HFHC_KO (hepatocyte PLD1-knockout mice with high-fat diet). Intestinal contents of mice are analyzed via metagenomics and metabolomics, and the liver bile acids are assessed by mass spectrometry imaging. The results show that at the phylum level the abundance of Bacillota in the intestines of MASLD model mice is significantly increased, whereas that of Bacteroidota significantly is decreased. However, after the deletion of hepatocyte PLD1, Pseudomonadota and Candidatus Bathyarchaeota are significantly decreased in the MASLD model mice. At the species level, compared with that in the Con group, the abundance of Faecalibaculum rodentium is significantly increased in the HFHC group, whereas hepatocyte PLD1 knockout causes the abundances of Desulfovibrionaceae bacterium LT0009 and Lachnospiraceae bacterium 10-1 to be significantly decreased. In terms of intestinal bile acids, the levels of two bile acids (hyodeoxycholic acid and glycolithocholic acid) differ between the HFHC_KO group and the HFHC group. Association analysis shows that Faecalibaculum co-occurs with DCA, βMCA, ΩMCA and αMCA, while probiotic Bacteroides uniformis is significantly correlated with UDCA, 12-KetoLCA, and 7-KetoLCA. Finally, mass spectrometry imaging reveals that the TCA and TDCA contents in the liver are significantly decreased after PLD1 knockout in hepatocytes. These findings demonstrate that hepatocyte PLD1 knockout alters the gut microbiota and bile acids profiles, suggesting that PLD1 deficiency may modulate MASLD progression by changing intestinal microbiota-bile acid homeostasis.

Amino acids (AAs) are closely linked to various diseases. Investigating their spatial distribution and content differences can provide deeper insights into specific disease mechanisms. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) enables spatial visualization of biomolecules, but conventional matrices introduce significant background interference that limits the detection of small molecules such as AAs. On-tissue chemical derivatization (OTCD) using permanently charged pyridinium probes significantly enhances the detection sensitivity of poorly ionizable compounds like AAs, allowing for their spatial mapping. However, current quantitative mass spectrometry imaging (QMSI) strategies for AAs using conventional matrix remain limited, highlighting the urgent need for the development of a widely applicable absolute quantification method for AAs that integrates OTCD. In this study, a series of pyridinium salt-based MALDI-MS probes were designed and characterized, leading to the identification of an efficient candidate, 1-(4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)-2-methylphenyl)-2,4,6-triphenylpyridin-1-ium tetrafluoroborate (DCMT-4FB). This probe was then combined with deuterium-labeled internal standard to establish calibration curve, and its linear correction capability was validated, demonstrating strong correlation coefficients. Furthermore, a novel quantitative endogenous substance spraying approach was employed to perform absolute quantification MSI analysis of AAs (leucine and isoleucine) in different regions of human hepatocellular carcinoma (HCC) tissue sections. Finally, by cospraying the DCMT-4FB probe with its deuterium-labeled isotope analog, DCMT-d2-4FB, the spatial distribution of AAs and other metabolites within HCC tissues was rapidly obtained, providing valuable insights for clinical research. This study highlights the superior AAs quantification capability of the DCMT-4FB probe and offers new perspectives for probe development and quantitative analysis of endogenous metabolites.

Unimodal Imaging of Monovalent Metal-Chelator Complexes and Lipids by MALDI Imaging Mass Spectrometry.