MALDI Imaging Research Articles

MALDI HiPLEX-IHC: multiomic and multimodal imaging of targeted intact proteins in tissues.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is one of the most widely used methods for imaging the spatial distribution of unlabeled small molecules such as metabolites, lipids and drugs in tissues. Recent progress has enabled many improvements including the ability to achieve single cell spatial resolution, 3D-tissue image reconstruction, and the precise identification of different isomeric and isobaric molecules. However, MALDI-MSI of high molecular weight intact proteins in biospecimens has thus far been difficult to achieve. Conventional methods normally require in situ proteolysis and peptide mass fingerprinting, have low spatial resolution, and typically detect only the most highly abundant proteins in an untargeted manner. In addition, MSI-based multiomic and multimodal workflows are needed which can image both small molecules and intact proteins from the same tissue. Such a capability can provide a more comprehensive understanding of the vast complexity of biological systems at the organ, tissue, and cellular levels of both normal and pathological function. A recently introduced top-down spatial imaging approach known as MALDI HiPLEX-IHC (MALDI-IHC for short) provides a basis for achieving this high-information content imaging of tissues and even individual cells. Based on novel photocleavable mass-tags conjugated to antibody probes, high-plex, multimodal and multiomic MALDI-based workflows have been developed to image both small molecules and intact proteins on the same tissue sample. Dual-labeled antibody probes enable multimodal mass spectrometry and fluorescent imaging of targeted intact proteins. A similar approach using the same photocleavable mass-tags can be applied to lectin and other probes. We detail here several examples of MALDI-IHC workflows designed to enable high-plex, multiomic and multimodal imaging of tissues at a spatial resolution as low as 5µm. This approach is compared to other existing high-plex methods such as imaging mass cytometry, MIBI-TOF, GeoMx and CODEX. Finally, future applications of MALDI-IHC are discussed.

Diverse organ-specific localisation of a chemical defence, cyanogenic glycosides, in flowers of eleven species of Proteaceae.

Floral chemical defence strategies remain under-investigated, despite the significance of flowers to plant fitness. We used cyanogenic glycosides (CNglycs)-constitutive secondary metabolites that deter herbivores by releasing hydrogen cyanide, but also play other metabolic roles-to ask whether more apparent floral tissues and those most important for fitness are more defended as predicted by optimal defence theories, and what fine-scale CNglyc localisation reveals about function(s)? Florets of eleven species from the Proteaceae family were dissected to quantitatively compare the distribution of CNglycs within flowers and investigate whether distributions vary with other floral/plant traits. CNglycs were identified and their localisation in florets was revealed by matrix-assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI). We identified extremely high CNglyc content in floral tissues of several species (>1% CN), highly tissue-specific CNglyc distributions within florets, and substantial interspecific differences in content distributions, not all consistent with optimal defence hypotheses. Four patterns of within-flower CNglyc allocation were identified: greater tissue-specific allocations to (1) anthers, (2) pedicel (and gynophore), (3) pollen presenter, and (4) a more even distribution among tissues with higher content in pistils. Allocation patterns were not correlated with other floral traits (e.g. colour) or taxonomic relatedness. MALDI-MSI identified differential localisation of two tyrosine-derived CNglycs, demonstrating the importance of visualising metabolite localisation, with the diglycoside proteacin in vascular tissues, and monoglycoside dhurrin across floral tissues. High CNglyc content, and diverse, specific within-flower localisations indicate allocations are adaptive, highlighting the importance of further research into the ecological and metabolic roles of floral CNglycs.

In Situ Proteomic Analysis of Herbicide-Resistant Soybean and Hybrid Seeds via Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging

Transgenic soybean is the commercial crop with the largest cultivation area worldwide. During transgenic soybean cultivation, exogenous genes may be transferred to wild relatives through gene flow, posing unpredictable ecological risks. Accordingly, an environmental risk assessment should focus on fitness changes and underlying mechanisms in hybrids between transgenic and wild soybeans (Glycine soja). Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) was used for in situ detection and imaging of protein changes in the seeds of transgenic herbicide-resistant soybean harboring epsps and pat genes, non-transgenic soybean, wild soybean, and their F2 hybrid. Protein data clearly distinguished wild soybeans, while the F2 seeds had protein characteristics of both parents and were distinguished from wild soybean seeds. Using UPLC-Q-TOF-MS, 22 differentially expressed proteins (DEPs) were identified, including 13 specific to wild soybean. Sucrose synthase and stress response-related DEPs were differentially expressed in parental and offspring. Differences in these may underpin the greater adaptability of the latter. MSI revealed DEP distribution in transgenic, wild, and F2 seeds. Identifying DEPs related to fitness may elucidate mechanisms underlying fitness differences among the studied varieties. Our study shows that MALDI-MSI has the potential to become a visual method for transgenic soybean analysis.

Mass Spectrometry Imaging in Alzheimer's Disease.

Introduction: Amyloid-beta (Aβ) pathology is the precipitating histopathological characteristic of Alzheimer's disease (AD). Although the formation of amyloid plaques in human brains is suggested to be a key factor in initiating AD pathogenesis, it is still not fully understood the upstream events that lead to Aβ plaque formation and its metabolism inside the brains. Methods: Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) has been successfully introduced to study AD pathology in brain tissue both in AD mouse models and human samples. By using MALDI-MSI, a highly selective deposition of Aβ peptides in AD brains with a variety of cerebral amyloid angiopathy (CAA) involvement was observed. Results: MALDI-MSI visualized depositions of shorter peptides in AD brains; Aβ1-36 to Aβ1-39 were quite similarly distributed with Aβ1-40 as a vascular pattern, and deposition of Aβ1-42 and Aβ1-43 was visualized with a distinct senile plaque pattern distributed in parenchyma. Moreover, how MALDI-MSI covered in situ lipidomics of plaque pathology has been reviewed, which is of interest as aberrations in neuronal lipid biochemistry have been implicated in AD pathogenesis. Discussion: In this study, we introduce the methodological concepts and challenges of MALDI-MSI for the studies of AD pathogenesis. Diverse Aβ isoforms including various C- and N-terminal truncations in AD and CAA brain tissues will be visualized. Despite the close relationship between vascular and plaque Aβ deposition, the current strategy will define cross talk between neurodegenerative and cerebrovascular processes at the level of Aβ metabolism.

In situ spatiotemporal mapping of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) inhibitor in pineapple (Ananas comosus) fruit tissue by MALDI mass spectrometry imaging

Pineapple (Ananas comosus) fruit is a functional food that has become a part of healthy diets worldwide due to its nutrition and health benefits. Given this concept, the potential anti-hypercholesterolemic activity of pineapple fruit was assessed using in vitro HMG-CoA reductase (HMGCR) inhibition assay. Our findings show that pineapple fruit (PAF) extract showed HMGCR inhibition activity. These findings led to partial purification of PAF extract by gel filtration chromatography (GFC) whereby fraction PAF 06 exhibited 100.00% HMGCR inhibition at 10 mg/mL. Matrix assisted laser desorption ionization (MALDI) mass spectrometry profiling, HRMS, MS/MS, and dereplication identified N1, N10-diferuloylspermidine as potential HMGCR inhibitor in pineapple fruit. The MALDI mass spectrometry imaging (MSI) enabled the in situ spatiotemporal mapping of the HMGCR inhibitor, N1, N10-diferuloylspermidine, in the pineapple fruit tissue, with localization concentrated mainly in the tissue near the peel/shell and a gradual decrease towards the flesh and ovary. The MALDI MSI provided the first evidence of in situ spatial localization of N1, N10-diferuloylspermidine, a potential HMGCR inhibitor in pineapple while maintaining the cellular and structural features of the fruit tissues.

On-Tissue Chemical Derivatization for Comprehensive Mapping of Brain Carboxyl and Aldehyde Metabolites by MALDI–MS Imaging

The visualization of small metabolites by MALDI mass spectrometry imaging in brain tissue sections is challenging due to low detection sensitivity and high background interference. We present an on-tissue chemical derivatization MALDI mass spectrometry imaging approach for the comprehensive mapping of carboxyls and aldehydes in brain tissue sections. In this approach, the AMPP (1-(4-(aminomethyl)phenyl)pyridin-1-ium chloride) derivatization reagent is used for the covalent charge-tagging of molecules containing carboxylic acid (in the presence of peptide coupling reagents) and aldehydes. This includes free fatty acids and the associated metabolites, fatty aldehydes, dipeptides, neurotoxic reactive aldehydes, amino acids, neurotransmitters and associated metabolites, as well as tricarboxylic acid cycle metabolites. We performed sensitive ultrahigh mass resolution MALDI-MS detection and imaging of various carboxyl- and aldehyde-containing endogenous metabolites simultaneously in rodent brain tissue sections. We verified the AMPP-derivatized metabolites by tandem MS for structural elucidation. This approach allowed us to image numerous aldehydes and carboxyls, including certain metabolites which had been undetectable in brain tissue sections. We also demonstrated the application of on-tissue derivatization to carboxyls and aldehydes in coronal brain tissue sections of a nonhuman primate Parkinson's disease model. Our methodology provides a powerful tool for the sensitive, simultaneous spatial molecular imaging of numerous aldehydes and carboxylic acids during pathological states, including neurodegeneration, in brain tissue.

Lipidomic Profiling of PFOA-Exposed Mouse Liver by Multi-Modal Mass Spectrometry Analysis.

Perfluorooctanoic acid (PFOA) is a synthetic perfluorinated chemical classified as a persistent organic pollutant. PFOA has been linked to many toxic effects, including liver injury. Many studies report that PFOA exposure alters serum and hepatic lipid metabolism. However, lipidomic pathways altered by PFOA exposure are largely unknown and only a few lipid classes, mostly triacylglycerol (TG), are usually considered in lipid analysis. Here, we performed a global lipidomic analysis on the liver of PFOA-exposed (high-dose and short-duration) and control mice by combining three mass spectrometry (MS) techniques: liquid chromatography with tandem mass spectrometry (LC-MS/MS), matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Among all hepatic lipids identified by LC-MS/MS analysis, more than 350 were statistically impacted (increased or decreased levels) after PFOA exposure, as confirmed by multi-variate data analysis. The levels of many lipid species from different lipid classes, most notably phosphatidylethanolamine (PE), phosphatidylcholine (PC), and TG, were significantly altered. Subsequent lipidomic analysis highlights the pathways significantly impacted by PFOA exposure, with the glycerophospholipid metabolism being the most impacted, and the changes in the lipidome network, which connects all the lipid species together. MALDI-MSI displays the heterogeneous distribution of the affected lipids and PFOA, revealing different areas of lipid expression linked to PFOA localization. TOF-SIMS localizes PFOA at the cellular level, supporting MALDI-MSI results. This multi-modal MS analysis unveils the lipidomic impact of PFOA in the mouse liver after high-dose and short-term exposure and opens new opportunities in toxicology.

In situ detection and mass spectrometry imaging of protein-related metabolites in Bombyx batryticatus before and after frying with wheat bran.

Bombyx batryticatus is derived from the dried larva of Bombyx mori Linnaeus infected by Beauveria bassiana (Bals.) Vuillant. Raw Bombyx batryticatus should be stir-fried before oral administration due to its irritation to the gastrointestinal tract. Nevertheless, it is still an arduous task to uncover the intrinsic mechanism of Bombyx batryticatus processing. In this study, we collected two types of Bombyx batryticatus, one being stir-fried and the other serving as a control. Then, an informative approach, which integrated matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) with chemometrics analysis, was established to screen processing-associated markers and reveal in situ spatial distribution patterns of protein-related metabolites. After optimization of experimental conditions, 21 ions were initially detected from Bombyx batryticatus, including amino acids and peptides. In addition, 15 differential markers were screened by orthogonal projection to potential structure discriminant analysis (OPLS-DA), which were localized and visualized in the transverse section of Bombyx batryticatus by MSI. Eventually, it can be demonstrated that the stir-frying process reduces toxicity while potentially boosting specific biological activities of Bombyx batryticatus. In summary, the established strategy could not only clarify the chemical transformation of protein-related metabolites from Bombyx batryticatus before and after frying with wheat bran, but also reveal the significance of Chinese medicine processing technology.

Tissue Microarray Lipidomic Imaging Mass Spectrometry Method: Application to the Study of Alcohol-Related White Matter Neurodegeneration.

Central nervous system (CNS) white matter pathologies accompany many diseases across the lifespan, yet their biochemical bases, mechanisms, and consequences have remained poorly understood due to the complexity of myelin lipid-based research. However, recent advances in matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) have minimized or eliminated many technical challenges that previously limited progress in CNS disease-based lipidomic research. MALDI-IMS can be used for lipid identification, semi-quantification, and the refined interpretation of histopathology. The present work illustrates the use of tissue micro-arrays (TMAs) for MALDI-IMS analysis of frontal lobe white matter biochemical lipidomic pathology in an experimental rat model of chronic ethanol feeding. The use of TMAs combines workload efficiency with the robustness and uniformity of data acquisition. The methods described for generating TMAs enable simultaneous comparisons of lipid profiles across multiple samples under identical conditions. With the methods described, we demonstrate significant reductions in phosphatidylinositol and increases in phosphatidylcholine in the frontal white matter of chronic ethanol-fed rats. Together with the use of a novel rapid peak alignment protocol, this approach facilitates reliable inter- and intra-group comparisons of MALDI-IMS data from experimental models and could be extended to human disease states, including using archival specimens.

Abstract 1993: Sulfobutylether-β-cyclodextrin hydrogel microspheres delivering TLR 7/8 agonist for transarterial immunoembolization

Abstract Drug-eluting bead transcatheter arterial chemoembolization (DEB-TACE) is a treatment procedure for liver cancer that involves the selective catheterization and subsequent embolization of tumor-feeding arteries with drug-eluting beads (DEBs). DEB-TACE elicits ischemic cell death in the embolized tumor while simultaneously delivering a local, sustained release of chemotherapy. We hypothesize that the application of DEBs loaded with an immunostimulatory adjuvant in the DEB-TACE procedure will promote local antigen presenting cells to utilize the antigens released by dying tumor cells to generate a systemic, adaptive anti-tumor immune response. This approach represents a novel form of transarterial immunoembolization (TIE). 558 is a highly potent, small molecule Toll-like receptor 7/8 agonist that activates both innate and adaptive immune responses to eliminate tumor cells in various preclinical tumor models. Hydrogel microspheres composed of cross-linked sulfobutylether-β-cyclodextrin (SBE-βCD) were investigated as DEBs for 558 in the current study. SBE-βCD hydrogel microspheres (SBE-βCDMS) of 10 - 300 μm diameter were synthesized via suspension polymerization of SBE-βCD and ethylene glycol diglycidyl ether followed by wet sieving. 558 loading was achieved by incubating blank SBE-βCDMS in aqueous solutions of 558. Under non-saturating conditions, SBE-βCDMS absorbed almost the entirety of 558 from loading solutions in 4 h. The dose of 558 loaded in SBE-βCDMS was tuned by altering the initial amount of 558 in solution, up to a maximum loading of 0.28 mg 558/mg dry SBE-βCDMS determined under saturating conditions. The time to 50% release of 558 from loaded SBE-βCDMS was less than 30 min when phosphate buffered saline was used as release media. However, the release of 558 was negligible when deionized water was used as release media. The released drug was as effective as free 558 in stimulating cytokine response from human peripheral blood mononuclear cells in vitro. As a surrogate for TIE, we evaluated plasma and tumor pharmacokinetics upon intratumoral injection of 558-loaded SBE-βCDMS (50 - 100 μm diameter) or free 558 at a dose of 100 μg in C57BL/6 mice bearing B16F10-OVA flank tumors. The gradual release of 558 from loaded SBE-βCDMS prevented an initial spike in plasma concentration that was observed for mice administered with free 558, and maintained constant tumor concentrations for at least 4 h post-injection. High-resolution MALDI mass spectrometry imaging of 15 μm-thick tumor cryosections indicated that 558 was initially concentrated within SBE-βCDMS after intratumoral injection, and extensively released into the surrounding tumor tissue 24 h post-injection. Taken together, these results suggest that 558-loaded SBE-βCDMS are a promising platform for local drug delivery and immune cell stimulation via TIE. Citation Format: Joel Updyke, Shubhmita Bhatnagar, Nitu Bhaskar, Rachel Parise, Swati Nagar, John Schultz, David Ferguson, Tamara Kucaba, Thomas Griffith, Ronald Siegel, Jayanth Panyam. Sulfobutylether-β-cyclodextrin hydrogel microspheres delivering TLR 7/8 agonist for transarterial immunoembolization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1993.

Autofocusing MALDI MS imaging of processed food exemplified by the contaminant acrylamide in German gingerbread

Acrylamide is a toxic reaction product occurring in dry-heated food such as bakery products. To meet the requirements laid down in recent international legal norms calling for reduction strategies in food prone to acrylamide formation, efficient chromatography-based quantification methods are available. However, for an efficient mitigation of acrylamide levels, not only the quantity, but also the contaminant’s distributions are of interest especially in inhomogeneous food consisting of multiple ingredients. A promising tool to investigate the spatial distribution of analytes in food matrices is mass spectrometry imaging (MS imaging). In this study, an autofocusing MALDI MS imaging method was developed for German gingerbread as an example for highly processed and instable food with uneven surfaces. Next to endogenous food constituents, the process contaminant acrylamide was identified and visualized keeping a constant laser focus throughout the measurement. Statistical analyses based on relative acrylamide intensities suggest a higher contamination of nut fragments compared to the dough. In a proof-of-concept experiment, a newly developed in-situ chemical derivatization protocol is described using thiosalicylic acid for highly selective detection of acrylamide. This study presents autofocusing MS imaging as a suitable complementary method for the investigation of analytes’ distributions in complex and highly processed food.

Stable isotope-labeled carnitine reveals its rapid transport into muscle cells and acetylation during contraction

Carnitine plays multiple roles in skeletal muscle metabolism, including fatty acid transport and buffering of excess acetyl-CoA in the mitochondria. The skeletal muscle cannot synthesize carnitine; therefore, carnitine must be taken up from the blood into the cytoplasm. Carnitine metabolism, its uptake into cells, and the subsequent reactions of carnitine are accelerated by muscle contraction. Isotope tracing enables the marking of target molecules and monitoring of tissue distribution. In this study, stable isotope-labeled carnitine tracing was combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging to determine carnitine distribution in mouse skeletal muscle tissues. Deuterium-labeled carnitine (d3-carnitine) was intravenously injected into the mice and diffused to the skeletal muscles for 30 and 60 min. To examine whether muscle contraction changes the distribution of carnitine and its derivatives, unilateral in situ muscle contraction was performed; 60 min muscle contraction showed increased d3-carnitine and its derivative d3-acetylcarnitine in the muscle, indicating that carnitine uptake in cells is promptly converted to acetylcarnitine, consequently, buffering accumulated acetyl-CoA. While the endogenous carnitine was localized in the slow type fibers rather than fast type, the contraction-induced distributions of d3-carnitine and acetylcarnitine were not necessarily associated with muscle fiber type. In conclusion, the combination of isotope tracing and MALDI-MS imaging can reveal carnitine flux during muscle contraction and show the significance of carnitine in skeletal muscles.

Impact of spatial metabolomics on immune-microenvironment in oral cancer prognosis: a clinical report.

MALDI imaging for metabolites and immunohistochemistry for 38 immune markers was used to characterize the spatial biology of 2 primary oral tumours, one from a patient with an early recurrence (Tumour R), and the other from a patient with no recurrence 2years after treatment completion (Tumour NR). Tumour R had an increased purine nucleotide metabolism in different regions of tumour and adenosine-mediated suppression of immune cells compared to Tumour NR. The differentially expressed markers in the different spatial locations in tumour R were CD33, CD163, TGF-β, COX2, PD-L1, CD8 and CD20. These results suggest that altered tumour metabolomics concomitant with a modified immune microenvironment could be a potential marker of recurrence.

MALDI Imaging Assisted Discovery of a Di-O-glycosyltransferase from Platycodon grandiflorum Root.

A matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) assisted genome mining strategy was developed for the discovery of glycosyltransferase (GT) from the root of Platycodon grandiflorum. A di-O-glycosyltransferase PgGT1 was discovered and characterized that is capable of catalyzing platycoside E (PE) synthesis through the attachment of two β-1,6-linked glucosyl residues sequentially to the glucosyl residue at the C3 position of platycodin D (PD). Although UDP-glucose is the preferred sugar donor for PgGT1, it could also utilize UDP-xylose and UDP-N-acetylglucosamine as weak donors. Residues S273, E274, and H350 played important roles in stabilizing the glucose donor and positioning the glucose in the optimal orientation for the glycosylation reaction. This study clarified two key steps involved in the biosynthetic pathway of PE and could greatly contribute to improving its industrial biotransformation.

A sensitive on-tissue chemical derivatization-mass spectrometry imaging method for the quantitative visualization of helicid in mice

Helicid (4-formylphenyl-O-β-d-allopyranoside) is an aromatic phenolic glucoside. It is one of the main active constituents from the seeds of the Chinese herb Helicia nilagirica. Helicid has been reported to exert sedative, analgesic, hypnotic and antidepressant effects, and is currently used clinically to treat neurasthenic syndrome, vascular headaches and trigeminal neuralgia. On-tissue chemical derivatization (OTCD) combined with matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) was developed for the quantitative visualization of helicid in mice. The derivatization reagent, Girard’s reagent T (GT), was sprayed directly onto the tissue surface using an automated pneumatic TM-sprayer. The OTCD reaction was conducted in a closed container with 120 mL of 50% methanol with 0.4% TFA at 40 °C for 40 min. α-Cyano-4-hydroxycinnamic acid (2.22 μg/mm2) was used as the matrix, which was deposited onto the tissue surface. MALDI-MSI analysis was performed in the positive-ion mode with a spatial resolution of 200 μm. The sensitivity for the MALDI-MSI of helicid in tissue sections increased 1920 times after derivatization. The assay showed good linearity in the helicid concentration range of 0.32–32 ng/mm2. The method was used to investigate the spatiotemporal distribution of helicid in mice.

MALDI-MSI of lipids in a model of breast cancer brain metastasis provides a surrogate measure of ischemia/hypoxia.

Breast cancer brain metastasis (BCBM) has an incidence of 10-30%. It is incurable and the biological mechanisms that promote its progression remain largely undefined. Consequently, to gain insights into BCBM processes, we have developed a spontaneous mouse model of BCBM and in this study found a 20% penetrance of macro-metastatic brain lesion formation. Considering that lipid metabolism is indispensable to metastatic progression, our goal was the mapping of lipid distributions throughout the metastatic regions of the brain. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) of lipids revealed that, relative to surrounding brain tissue, seven long-chain (13-21 carbons long) fatty acylcarnitines, as well as two phosphatidylcholines, two phosphatidylinositols two diacylglycerols, a long-chain phosphatidylethanolamine, and a long-chain sphingomyelin were highly concentrated in the metastatic brain lesion In broad terms, lipids known to be enriched in brain tissues, such as very long-chain (≥ 22 carbons in length) polyunsaturated fatty acid of phosphatidylcholines, phosphatidylethanolamine, sphingomyelins, sulfatides, phosphatidylinositol phosphates, and galactosylceramides, were not found or only found in trace amounts in the metastatic lesion and instead consistently detected in surrounding brain tissues. The data, from this mouse model, highlights an accumulation of fatty acylcarnitines as possible biological makers of a chaotic inefficient vasculature within the metastasis, resulting in relatively inadequate blood flow and disruption of fatty acid β-oxidation due to ischemia/hypoxia.

Visualizing the distribution of flavonoids in litchi (Litchi chinenis) seeds through matrix-assisted laser desorption/ionization mass spectrometry imaging.

Flavonoids are one of the most important bioactive components in litchi (Litchi chinensis Sonn.) seeds and have broad-spectrum antiviral and antitumor activities. Litchi seeds have been shown to inhibit the proliferation of cancer cells and induce apoptosis, particularly effective against breast and liver cancers. Elucidating the distribution of flavonoids is important for understanding their physiological and biochemical functions and facilitating their efficient extraction and utilization. However, the spatial distribution patterns and expression states of flavonoids in litchi seeds remain unclear. Herein, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used for in situ detection and imaging of the distribution of flavonoids in litchi seed tissue sections for the first time. Fifteen flavonoid ion signals, including liquiritigenin, apigenin, naringenin, luteolin, dihydrokaempferol, daidzein, quercetin, taxifolin, kaempferol, isorhamnetin, myricetin, catechin, quercetin 3-β-d-glucoside, baicalin, and rutin, were successfully detected and imaged in situ through MALDI-MSI in the positive ion mode using 2-mercaptobenzothiazole as a matrix. The results clearly showed the heterogeneous distribution of flavonoids, indicating the potential of litchi seeds for flavonoid compound extraction. MALDI-MS-based multi-imaging enhanced the visualization of spatial distribution and expression states of flavonoids. Thus, apart from improving our understanding of the spatial distribution of flavonoids in litchi seeds, our findings also facilitate the development of MALDI-MSI-based metabolomics as a novel effective molecular imaging tool for evaluating the spatial distribution of endogenous compounds.

Spatial resolution of renal amyloid deposits through MALDI-MSI: a combined digital and molecular approach to monoclonal gammopathies

AimsIdentification and characterisation of monoclonal gammopathies of renal significance (MGRS) is critical for therapeutic purposes. Amyloidosis represents one of the most common forms of MGRS, and renal biopsy remains the...

Spatial analysis of the ancient proteome of archeological teeth using mass spectrometry imaging.

Proteins extracted from archaeological bone and teeth are utilised for investigating the phylogeny of extinct and extant species, the biological sex and age of past individuals, as well as ancient health and physiology. However, variable preservation of proteins in archaeological materials represents a major challenge. To better understand the spatial distribution of ancient proteins preserved within teeth, we applied matrix assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) for the first time to bioarchaeological samples to visualise the intensity of proteins in archaeological teeth thin sections. We specifically explored the spatial distribution of four proteins (collagen type I, of whichthe chains alpha-1 and alpha-2, alpha-2-HS-glycoprotein, haemoglobin subunit alpha and myosin light polypeptide 6). We successfully identified ancient proteins in archaeological teeth thin sections using mass spectrometry imaging. The data are available via ProteomeXchange with identifier PXD038114. However, we observed that peptides did not always follow our hypotheses for their spatial distribution, with distinct differences observed in the spatial distribution of several proteins, and occasionally between peptides of the same protein. While it remains unclear what causes these differences in protein intensity distribution within teeth, as revealed by MALDI-MSI in this study, we have demonstrated that MALDI-MSI can be successfully applied to mineralised bioarchaeological tissues to detect ancient peptides. In future applications, this technique could be particularly fruitful not just for understanding the preservation of proteins in a range of archaeological materials, but making informed decisions on sampling strategies and the targeting of key proteins of archaeological and biological interest.

Is a Modified Actin the Key to Toxin Resistance in the Nudibranch Chromodoris? A Biochemical and Molecular Approach

Five Chromodoris species from North Sulawesi, Indonesia, were investigated for their sequestration of marine natural products. The cytotoxic 2-thiazolidinone macrolide latrunculin A (LatA) was the major metabolite in all examined Chromodoris species, as well as in one of the associated sponges Cacospongia mycofijiensis (Kakou, Crews & Bakus, 1987), supporting a dietary origin of LatA. Furthermore, LatA was secreted with the mucus trail, suggesting a possible use in short-range chemical communication. MALDI MS-Imaging revealed an accumulation of LatA throughout the mantle tissue, mucus glands, and especially in vacuoles of the mantle dermal formations (MDFs). Cytotoxicity of the isolated LatA was tested in HEK-293 cells, confirming that LatA targets the actin cytoskeleton. In vivo toxicity experiments with the sacoglossan Elysia viridis (Montagu, 1804) showed 100% mortality, but 100% survival of Chromodoris specimens, demonstrating resistance to LatA. A novel actin isoform was detected in all investigated Chromodoris species with two amino acid substitutions at the ‘nucleotide binding’ cleft, the binding site of LatA. These are suggested to cause insensitivity against LatA, thus enabling the storage of the toxin within the body for the slugs’ own defense.