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BACKGROUND: The metabolic alterations occurring within the arterial architecture during atherosclerosis development remain poorly understood, let alone those particular to each arterial tunica. We aimed first to identify, in a spatially resolved manner, the specific metabolic changes in plaque, media, adventitia, and cardiac tissue between control and atherosclerotic murine aortas. Second, we assessed their translatability to human tissue and plasma for cardiovascular risk estimation. METHODS: In this observational study, mass spectrometry imaging (MSI) was applied to identify region-specific metabolic differences between atherosclerotic (n=11) and control (n=11) aortas from low-density lipoprotein receptor-deficient mice, via histology-guided virtual microdissection. Early and advanced plaques were compared within the same atherosclerotic animals. Progression metabolites were further analyzed by MSI in 9 human atherosclerotic carotids and by targeted mass spectrometry in human plasma from subjects with elective coronary artery bypass grafting (cardiovascular risk group, n=27) and a control group (n=27). RESULTS: MSI identified 362 local metabolic alterations in atherosclerotic mice (log2 fold-change ≥1.5; P≤0.05). The lipid composition of cardiac tissue is altered during atherosclerosis development and presents a generalized accumulation of glycerophospholipids, except for lysolipids. Lysolipids (among other glycerophospholipids) were found at elevated levels in all 3 arterial layers of atherosclerotic aortas. LPC(18:0) (lysophosphatidylcholine; P=0.024) and LPA(18:1) (lysophosphatidic acid; P=0.025) were found to be significantly elevated in advanced plaques as compared with mouse-matched early plaques. Higher levels of both lipid species were also observed in fibrosis-rich areas of advanced- versus early-stage human samples. They were found to be significantly reduced in human plasma from subjects with elective coronary artery bypass grafting (P<0.001 and P=0.031, respectively), with LPC(18:0) showing significant association with cardiovascular risk (odds ratio, 0.479 [95% CI, 0.225-0.883]; P=0.032) and diagnostic potential (area under the curve, 0.778 [95% CI, 0.638-0.917]). CONCLUSIONS: An altered phospholipid metabolism occurs in atherosclerosis, affecting both the aorta and the adjacent heart tissue. Plaque-progression lipids LPC(18:0) and LPA(18:1), as identified by MSI on tissue, reflect cardiovascular risk in human plasma.
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Doenças da Aorta , Aterosclerose , Doenças Cardiovasculares , Placa Aterosclerótica , Humanos , Animais , Camundongos , Placa Aterosclerótica/metabolismo , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/metabolismo , Fatores de Risco , Aterosclerose/diagnóstico , Aterosclerose/metabolismo , Aorta/diagnóstico por imagem , Aorta/metabolismo , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Glicerofosfolipídeos/metabolismo , Fatores de Risco de Doenças CardíacasRESUMO
Focal cortical dysplasia (FCD) represents a group of diverse localized cortical lesions that are highly epileptogenic and occur due to abnormal brain development caused by genetic mutations, involving the mammalian target of rapamycin (mTOR). These somatic mutations lead to mosaicism in the affected brain, posing challenges to unravel the direct and indirect functional consequences of these mutations. To comprehensively characterize the impact of mTOR mutations on the brain, we employed here a multimodal approach in a preclinical mouse model of FCD type II (Rheb), focusing on spatial omics techniques to define the proteomic and lipidomic changes. Mass Spectrometry Imaging (MSI) combined with fluorescence imaging and label free proteomics, revealed insight into the brain's lipidome and proteome within the FCD type II affected region in the mouse model. MSI visualized disrupted neuronal migration and differential lipid distribution including a reduction in sulfatides in the FCD type II-affected region, which play a role in brain myelination. MSI-guided laser capture microdissection (LMD) was conducted on FCD type II and control regions, followed by label free proteomics, revealing changes in myelination pathways by oligodendrocytes. Surgical resections of FCD type IIb and postmortem human cortex were analyzed by bulk transcriptomics to unravel the interplay between genetic mutations and molecular changes in FCD type II. Our comparative analysis of protein pathways and enriched Gene Ontology pathways related to myelination in the FCD type II-affected mouse model and human FCD type IIb transcriptomics highlights the animal model's translational value. This dual approach, including mouse model proteomics and human transcriptomics strengthens our understanding of the functional consequences arising from somatic mutations in FCD type II, as well as the identification of pathways that may be used as therapeutic strategies in the future.
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Epilepsia , Malformações do Desenvolvimento Cortical do Grupo I , Proteômica , Animais , Humanos , Malformações do Desenvolvimento Cortical do Grupo I/genética , Malformações do Desenvolvimento Cortical do Grupo I/metabolismo , Malformações do Desenvolvimento Cortical do Grupo I/patologia , Camundongos , Masculino , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Feminino , Modelos Animais de Doenças , Encéfalo/metabolismo , Encéfalo/patologia , Proteoma/metabolismo , Displasia Cortical FocalRESUMO
We introduce a novel approach for comprehensive molecular profiling in biological samples. Our single-section methodology combines quantitative mass spectrometry imaging (Q-MSI) and a single step extraction protocol enabling lipidomic and proteomic liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis on the same tissue area. The integration of spatially correlated lipidomic and proteomic data on a single tissue section allows for a comprehensive interpretation of the molecular landscape. Comparing Q-MSI and Q-LC-MS/MS quantification results sheds new light on the effect of MSI and related sample preparation. Performing MSI before Q-LC-MS on the same tissue section led to fewer protein identifications and a lower correlation between lipid quantification results. Also, the critical role and influence of internal standards in Q-MSI for accurate quantification is highlighted. Testing various slide types and the evaluation of different workflows for single-section spatial multiomics analysis emphasized the need for critical evaluation of Q-MSI data. These findings highlight the necessity for robust quantification methods comparable to current gold-standard LC-MS/MS techniques. The spatial information from MSI allowed region-specific insights within heterogeneous tissues, as demonstrated for glioblastoma multiforme. Additionally, our workflow demonstrated the efficiency of a single step extraction for lipidomic and proteomic analyses on the same tissue area, enabling the examination of significantly altered proteins and lipids within distinct regions of a single section. The integration of these insights into a lipid-protein interaction network expands the biological information attainable from a tissue section, highlighting the potential of this comprehensive approach for advancing spatial multiomics research.
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Lipidômica , Espectrometria de Massas em Tandem , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cromatografia Líquida , Fluxo de Trabalho , Espectrometria de Massa com Cromatografia Líquida , Proteômica/métodos , Lipídeos/análiseRESUMO
Matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) is a powerful analytical tool that enables molecular sample analysis while simultaneously providing the spatial context of hundreds or even thousands of analytes. However, because of the lack of a separation step prior to ionization and the immense diversity of biomolecules, such as lipids, including numerous isobaric species, the coupling of ultrahigh mass resolution (UHR) with MSI presents one way in which this complexity can be resolved at the spectrum level. Until now, UHR MSI platforms have been restricted to Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Here, we demonstrate the capabilities of an Orbitrap-based UHR MSI platform to reach over 1,000,000 mass resolution in a lipid mass range (600-950 Da). Externally coupling the Orbitrap Q Exactive HF with the high-performance data acquisition system FTMS Booster X2 provided access to the unreduced data in the form of full-profile absorption-mode FT mass spectra. In addition, it allowed us to increase the time-domain transient length from 0.5 to 10 s, providing improvement in the mass resolution, signal-to-noise ratio, and mass accuracy. The resulting UHR performance generates high-quality MALDI MSI images and simplifies the identification of lipids. Collectively, these improvements resulted in a 1.5-fold increase in annotations, demonstrating the advantages of this UHR imaging platform for spatial lipidomics using MALDI-MSI.
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Ciclotrons , Diagnóstico por Imagem , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Análise de Fourier , Lipídeos/análiseRESUMO
Aquatic ferns of the genus Azolla (Azolla) form highly productive symbioses with filamentous cyanobacteria fixing N2 in their leaf cavities, Nostoc azollae. Stressed symbioses characteristically turn red due to 3-deoxyanthocyanidin (DA) accumulation, rare in angiosperms and of unknown function. To understand DA accumulation upon cold acclimation and recovery, we integrated laser-desorption-ionization mass-spectrometry-imaging (LDI-MSI), a new Azolla filiculoides genome-assembly and annotation, and dual RNA-sequencing into phenotypic analyses of the symbioses. Azolla sp. Anzali recovered even when cold-induced DA-accumulation was inhibited by abscisic acid. Cyanobacterial filaments generally disappeared upon cold acclimation and Nostoc azollae transcript profiles were unlike those of resting stages formed in cold-resistant sporocarps, yet filaments re-appeared in leaf cavities of newly formed green fronds upon cold-recovery. The high transcript accumulation upon cold acclimation of AfDFR1 encoding a flavanone 4-reductase active in vitro suggested that the enzyme of the first step in the DA-pathway may regulate accumulation of DAs in different tissues. However, LDI-MSI highlighted the necessity to describe metabolite accumulation beyond class assignments as individual DA and caffeoylquinic acid metabolites accumulated differentially. For example, luteolinidin accumulated in epithelial cells, including those lining the leaf cavity, supporting a role for the former in the symbiotic interaction during cold acclimation.
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BACKGROUND: Soft tissue sarcomas (STS) constitute a heterogeneous group of rare tumor entities. Treatment relies on challenging patient-tailored surgical resection. Real-time intraoperative lipid profiling of electrosurgical vapors by rapid evaporative ionization mass spectrometry (REIMS) may aid in achieving successful surgical R0 resection (i.e., microscopically negative-tumor margin resection). Here, we evaluate the ex vivo accuracy of REIMS to discriminate and identify various STS from normal surrounding tissue. METHODS: Twenty-seven patients undergoing surgery for STS at Maastricht University Medical Center+ were included in the study. Samples of resected STS specimens were collected and analyzed ex vivo using REIMS. Electrosurgical cauterization of tumor and surrounding was generated successively in both cut and coagulation modes. Resected specimens were subsequently processed for gold standard histopathological review. Multivariate statistical analysis (principal component analysis-linear discriminant analysis) and leave-one patient-out cross-validation were employed to compare the classifications predicted by REIMS lipid profiles to the pathology classifications. Electrosurgical vapors produced during sarcoma resection were analyzed in vivo using REIMS. RESULTS: In total, 1200 histopathologically-validated ex vivo REIMS lipid profiles were generated from 27 patients. Ex vivo REIMS lipid profiles classified STS and normal tissues with 95.5% accuracy. STS, adipose and muscle tissues were classified with 98.3% accuracy. Well-differentiated liposarcomas and adipose tissues could not be discriminated based on their respective lipid profiles. Distinction of leiomyosarcomas from other STS could be achieved with 96.6% accuracy. In vivo REIMS analyses generated intense mass spectrometric signals. CONCLUSION: Lipid profiling by REIMS is able to discriminate and identify STS with high accuracy and therefore constitutes a potential asset to improve surgical resection of STS in the future.
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Sarcoma , Neoplasias de Tecidos Moles , Humanos , Eletrocirurgia/métodos , Sarcoma/cirurgia , Espectrometria de Massas/métodos , Neoplasias de Tecidos Moles/cirurgia , Margens de Excisão , LipídeosRESUMO
RATIONALE: Matrix-assisted laser desorption/ionisation-mass spectrometry imaging (MALDI-MSI) is a powerful label-free technique for biomolecule detection (e.g., lipids), within tissue sections across various biological species. However, despite its utility in many applications, the nematode Caenorhabditis elegans is not routinely used in combination with MALDI-MSI. The lack of studies exploring spatial distribution of biomolecules in nematodes is likely due to challenges with sample preparation. METHODS: This study developed a sample preparation method for whole intact nematodes, evaluated using cryosectioning of nematodes embedded in a 10% gelatine solution to obtain longitudinal cross sections. The slices were then subjected to MALDI-MSI, using a RapifleX Tissuetyper in positive and negative polarities. Samples were also prepared for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using an Exploris 480 coupled to a HPLC Vanquish system to confirm the MALDI-MSI results. RESULTS: An optimised embedding method was developed for longitudinal cross-sectioning of individual worms. To obtain longitudinal cross sections, nematodes were frozen at -80°C so that all worms were rod shaped. Then, the samples were defrosted and transferred to a 10% gelatine matrix in a cryomold; the worms aligned, and the whole cryomold submerged in liquid nitrogen. Using MALDI-MSI, we were able to observe the distribution of lipids within C. elegans, with clear differences in their spatial distribution at a resolution of 5 µm. To confirm the lipids from MALDI-MSI, age-matched nematodes were subjected to LC-MS/MS. Here, 520 lipids were identified using LC-MS/MS, indicating overlap with MALDI-MSI data. CONCLUSIONS: This optimised sample preparation technique enabled (un)targeted analysis of spatially distributed lipids within individual nematodes. The possibility to detect other biomolecules using this method thus laid the basis for prospective preclinical and toxicological studies on C. elegans.
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Caenorhabditis elegans , Lipídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Animais , Caenorhabditis elegans/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodos , Lipídeos/análise , Lipídeos/química , Cromatografia Líquida/métodosRESUMO
The spatial distribution of molecules and compounds responsible for the flavor profile of edible button mushrooms (Agaricus bisporous) has never been determined. The food industry is interested in knowing the localization of these compounds. Such knowledge would enable extraction of flavor compounds from a particular regions of the mushroom, which is safer for consumption compared to alternatives such as synthetic flavoring agents. The present study utilizes matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI), to determine the spatial distribution of flavor compounds in a mushroom. As MALDI-MSI requires very thin sections, a sample preparation protocol was optimized and sectioning fresh frozen mushrooms at 35 µm thickness was considered the best method to evaluate the distribution of flavor compounds. Further, the effect of heat on the spatial distribution of flavor compounds was investigated by heating whole mushrooms to 140 â prior to sectioning. Heating reduced the water content of the mushroom and thus enabled the generation of even-thinner 17 µm thick sections. MALDI-MSI measurements performed on underivatized and on-tissue derivatized fresh frozen and heat-treated mushroom sections elucidated the spatial distribution of several flavor-related compounds. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05883-0.
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BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and leads to the poorest patient outcomes despite surgery and chemotherapy treatment. Exploring new molecular mechanisms of TNBC that could lead to the development of novel molecular targets are critically important for improving therapeutic options for treating TNBC. METHODS: We sought to identify novel therapeutic targets in TNBC by combining genomic and functional studies with lipidomic analysis, which included mechanistic studies to elucidate the pathways that tie lipid profile to critical cancer cell properties. Our studies were performed in a large panel of human breast cancer cell lines and patient samples. RESULTS: Comprehensive lipid profiling revealed that phospholipid metabolism is reprogrammed in TNBC cells. We discovered that patatin-like phospholipase domain-containing lipase 8 (PNPLA8) is overexpressed in TNBC cell lines and tissues from breast cancer patients. Silencing of PNPLA8 disrupted phospholipid metabolic reprogramming in TNBC, particularly affecting the levels of phosphatidylglycerol (PG), phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and glycerophosphocholine (GPC). We showed that PNPLA8 is essential in regulating cell viability, migration and antioxidation in TNBC cells and promoted arachidonic acid and eicosanoid production, which in turn activated PI3K/Akt/Gsk3ß and MAPK signaling. CONCLUSIONS: Our study highlights PNPLA8 as key regulator of phospholipid metabolic reprogramming and malignant phenotypes in TNBC, which could be further developed as a novel molecular treatment target.
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Neoplasias de Mama Triplo Negativas , Humanos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Fenótipo , Fosfatidilinositol 3-Quinases/genética , Fosfolipídeos/uso terapêutico , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The Timepix (TPX) is a position- and time-sensitive pixelated charge detector that can be coupled with time-of-flight mass spectrometry (TOF MS) in combination with microchannel plates (MCPs) for the spatially and temporally resolved detection of biomolecules. Earlier generation TPX detectors used in previous studies were limited by a moderate time resolution (at best 10 ns) and single-stop detection for each pixel that hampered the detection of ions with high mass-to-charge (m/z) values at high pixel occupancies. In this study, we have coupled an MCP-phosphor screen-TPX3CAM detection assembly that contains a silicon-coated TPX3 chip to a matrix-assisted laser desorption/ionization (MALDI)-axial TOF MS. A time resolution of 1.5625 ns, per-pixel multihit functionality, simultaneous measurement of TOF and time-over-threshold (TOT) values, and kHz readout rates of the TPX3 extended the m/z detection range of the TPX detector family. The detection of singly charged intact Immunoglobulin M ions of m/z value approaching 1 × 106 Da has been demonstrated. We also discuss the utilization of additional information on impact coordinates and TOT provided by the TPX3 compared to conventional MS detectors for the enhancement of the quality of the mass spectrum in terms of signal-to-noise (S/N) ratio. We show how the reduced dead time and event-based readout in TPX3 compared to the TPX improves the sensitivity of high m/z detection in both low and high mass measurements (m/z range: 757-970,000 Da). We further exploit the imaging capabilities of the TPX3 detector for the spatial and temporal separation of neutral fragments generated by metastable decay at different locations along the field-free flight region by simultaneous application of deflection and retarding fields.
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Diagnóstico por Imagem , Silício , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Íons , LasersRESUMO
Recently, a novel technology was published, utilizing the strengths of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) and immunohistochemistry (IHC), achieving highly multiplexed, targeted imaging of biomolecules in tissue. This new technique, called MALDI-IHC, opened up workflows to target molecules of interest using MALDI-MSI that are usually targeted by standard IHC. In this paper, the utility of targeted MALDI-IHC and its complementarity with untargeted on-tissue bottom-up spatial proteomics is explored using breast cancer tissue. Furthermore, the MALDI-2 effect was investigated and demonstrated to improve MALDI-IHC. Formalin-fixed paraffin-embedded (FFPE) human breast cancer tissue sections were stained for multiplex MALDI-IHC with six photocleavable mass-tagged (PC-MT) antibodies constituting a breast cancer antibody panel (CD20, actin-αSM, HER2, CD68, vimentin, and panCK). K-means spatial clusters were created based on the MALDI-IHC images and cut out using laser-capture microdissection (LMD) for further untargeted LC-MS-based bottom-up proteomics analyses. Numerous peptides could be tentatively assigned to multiple proteins, of which three proteins were also part of the antibody panel (vimentin, keratins, and actin). Post-ionization with MALDI-2 showed an increased intensity of the PC-MTs and suggests options for the development of new mass-tags. Although the on-tissue digestion covered a wider range of proteins, the MALDI-IHC allowed for easy and straightforward identification of proteins that were not detected in untargeted approaches. The combination of the multiplexed MALDI-IHC with image-guided proteomics showed great potential to further investigate diseases by providing complementary information from the same tissue section and without the need for customized instrumentation.
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Neoplasias da Mama , Proteômica , Humanos , Feminino , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Vimentina , Proteômica/métodos , Imuno-Histoquímica , Actinas , Imagem MolecularRESUMO
In the past decade, interest in organoids for biomedical research has surged, resulting in a higher demand for advanced imaging techniques. Traditional specimen embedding methods pose challenges, such as analyte delocalization and histological assessment. Here, we present an optimized sample preparation approach utilizing an Epredia M-1 cellulose-based embedding matrix, which preserves the structural integrity of fragile small intestinal organoids (SIOs). Additionally, background interference (delocalization of analytes, nonspecific (histological) staining, matrix ion clusters) was minimized, and we demonstrate the compatibility with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). With our approach, we can conduct label-free lipid imaging at the single-cell level, thereby yielding insights into the spatial distribution of lipids in both positive and negative ion modes. Moreover, M-1 embedding allows for an improved coregistration with histological and immunohistochemical (IHC) stainings, including MALDI-IHC, facilitating combined untargeted and targeted spatial information. Applying this approach, we successfully phenotyped crypt-like (CL) and villus-like (VL) SIOs, revealing that PE 36:2 [M - H]- (m/z 742.5) and PI 38:4 [M - H]- (m/z 885.5) display higher abundance in CL organoids, whereas PI 36:1 [M - H]- (m/z 863.6) was more prevalent in VL organoids. Our findings demonstrate the utility of M-1 embedding for advancing organoid research and unraveling intricate biological processes within these in vitro models.
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Diagnóstico por Imagem , Lipidômica , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Manejo de Espécimes , LasersRESUMO
Mass spectrometry imaging (MSI) has accelerated our understanding of lipid metabolism and spatial distribution in tissues and cells. However, few MSI studies have approached lipid imaging quantitatively and those that have focused on a single lipid class. We overcome this limitation by using a multiclass internal standard (IS) mixture sprayed homogeneously over the tissue surface with concentrations that reflect those of endogenous lipids. This enabled quantitative MSI (Q-MSI) of 13 lipid classes and subclasses representing almost 200 sum-composition lipid species using both MALDI (negative ion mode) and MALDI-2 (positive ion mode) and pixel-wise normalization of each lipid species in a manner analogous to that widely used in shotgun lipidomics. The Q-MSI approach covered 3 orders of magnitude in dynamic range (lipid concentrations reported in pmol/mm2) and revealed subtle changes in distribution compared to data without normalization. The robustness of the method was evaluated by repeating experiments in two laboratories using both timsTOF and Orbitrap mass spectrometers with an â¼4-fold difference in mass resolution power. There was a strong overall correlation in the Q-MSI results obtained by using the two approaches. Outliers were mostly rationalized by isobaric interferences or the higher sensitivity of one instrument for a particular lipid species. These data provide insight into how the mass resolving power can affect Q-MSI data. This approach opens up the possibility of performing large-scale Q-MSI studies across numerous lipid classes and subclasses and revealing how absolute lipid concentrations vary throughout and between biological tissues.
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Diagnóstico por Imagem , Lipidômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Lipídeos/análise , Encéfalo/metabolismoRESUMO
Mass spectrometry imaging (MSI) combines molecular and spatial information in a valuable tool for a wide range of applications. Matrix-assisted laser desorption/ionization (MALDI) is at the forefront of MSI ionization due to its wide availability and increasing improvement in spatial resolution and analysis speed. However, ionization suppression, low concentrations, and endogenous and methodological interferences cause visualization problems for certain molecules. Chemical derivatization (CD) has proven a viable solution to these issues when applied in mass spectrometry platforms. Chemical tagging of target analytes with larger, precharged moieties aids ionization efficiency and removes analytes from areas of potential isobaric interferences. Here, we address the application of CD on tissue samples for MSI analysis, termed on-tissue chemical derivatization (OTCD). MALDI MSI will remain the focus platform due to its popularity, however, alternative ionization techniques such as liquid extraction surface analysis and desorption electrospray ionization will also be recognized. OTCD reagent selection, application, and optimization methods will be discussed in detail. MSI with OTCD is a powerful tool to study the spatial distribution of poorly ionizable molecules within tissues. Most importantly, the use of OTCD-MSI facilitates the analysis of previously inaccessible biologically relevant molecules through the adaptation of existing CD methods. Though further experimental optimization steps are necessary, the benefits of this technique are extensive.
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Processamento de Imagem Assistida por Computador , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Mass spectrometry imaging (MSI) is a powerful technique enabling the visualization of the spatial distribution of different molecules in tissue biopsies with different pathologies. Sample handling and preparing adipose tissue for MSI is challenging and prone to molecular delocalization due to tissue melting. In this work, we developed a method for matrix-assisted laser desorption/ionization (MALDI)-MSI to study lipids in human infrapatellar fat pad (IPFP), a biomarker source in musculoskeletal pathologies, while preserving molecular spatial distribution. Cryosectioning at 15 µm with a temperature below -30 °C, thaw-mounting, and sublimation, was demonstrated to preserve IPFP's heterogeneous appearance and spatial distribution of lipids.
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Diagnóstico por Imagem , Manejo de Espécimes , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Lipídeos/análise , LasersRESUMO
Human macrophages are innate immune cells with diverse, functionally distinct phenotypes, namely, pro-inflammatory M1 and anti-inflammatory M2 macrophages. Both are involved in multiple physiological and pathological processes, including would healing, infection, and cancer. However, the metabolic differences between these phenotypes are largely unexplored at single-cell resolution. To address this knowledge gap, an untargeted live single-cell mass spectrometry-based metabolomic profiling coupled with a machine-learning data analysis approach was developed to investigate the metabolic profile of each phenotype at the single-cell level. Results show that M1 and M2 macrophages have distinct metabolic profiles, with differential levels of fatty acyls, glycerophospholipids, and sterol lipids, which are important components of plasma membrane and involved in multiple biological processes. Furthermore, we could discern several putatively annotated molecules that contribute to inflammatory response of macrophages. The combination of random forest and live single-cell metabolomics provided an in-depth profile of the metabolome of primary human M1 and M2 macrophages at the single-cell level for the first time, which will pave the way for future studies targeting the differentiation of other immune cells.
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Macrófagos , Algoritmo Florestas Aleatórias , Humanos , Macrófagos/metabolismo , Metabolômica , Metaboloma , FenótipoRESUMO
RATIONALE: Isomeric separation of prostanoids is often a challenge and requires chromatography and time-consuming sample preparation. Multiple prostanoid isomers have distinct in vivo functions crucial for understanding the inflammation process, including prostaglandins E2 (PGE2 ) and D2 (PGD2 ). High-resolution ion mobility spectrometry (IMS) based on linear ion transport in low-to-moderate electric fields and nonlinear ion transport in strong electric fields emerges as a broad approach for rapid separations prior to mass spectrometry. METHODS: Derivatization with Girard's reagent T (GT) was used to overcome inefficient ionization of prostanoids in negative ionization mode due to poor deprotonation of the carboxylic acid group. Three high-resolution IMS techniques, namely linear cyclic IMS, linear trapped IMS, and nonlinear high-field asymmetric waveform IMS, were compared for the isomeric separation and endogenous detection of prostanoids present in intestinal tissue. RESULTS: Direct infusion of GT-derivatized prostanoids proved to increase the ionization efficiency in positive ionization mode by a factor of >10, which enabled detection of these molecules in endogenous concentration levels. The high-resolution IMS comparison revealed its potential for rapid isomeric analysis of biologically relevant prostanoids. Strengths and weaknesses of both linear and nonlinear IMS are discussed. Endogenous prostanoid detection in intestinal tissue extracts demonstrated the applicability of our approach in biomedical research. CONCLUSIONS: The applied derivatization strategy offers high sensitivity and improved stereoisomeric separation for screening of complex biological systems. The high-resolution IMS comparison indicated that the best sensitivity and resolution are achieved by linear and nonlinear IMS, respectively.
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Espectrometria de Mobilidade Iônica , Prostaglandinas , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos , Betaína/químicaRESUMO
Mass spectrometry imaging has advanced from a niche technique to a widely applied spatial biology tool operating at the forefront of numerous fields, most notably making a significant impact in biomedical pharmacological research. The growth of the field has gone hand in hand with an increase in publications and usage of the technique by new laboratories, and consequently this has led to a shift from general MSI reviews to topic-specific reviews. Given this development, we see the need to recapitulate the strengths of MSI by providing a more holistic overview of state-of-the-art MSI studies to provide the new generation of researchers with an up-to-date reference framework. Here we review scientific advances for the six largest biomedical fields of MSI application (oncology, pharmacology, neurology, cardiovascular diseases, endocrinology, and rheumatology). These publications thereby give examples for at least one of the following categories: they provide novel mechanistic insights, use an exceptionally large cohort size, establish a workflow that has the potential to become a high-impact methodology, or are highly cited in their field. We finally have a look into new emerging fields and trends in MSI (immunology, microbiology, infectious diseases, and aging), as applied MSI is continuously broadening as a result of technological breakthroughs.
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Pesquisa Biomédica , Diagnóstico por Imagem , Humanos , Espectrometria de Massas/métodos , Diagnóstico por Imagem/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
The incidence of osteoarthritis (OA) has been expected to increase due to an aging population, as well as an increased incidence of intra-articular (osteo-) chondral damage. Lipids have already been shown to be involved in the inflammatory process of OA. This study aims at revealing region-specific lipid profiles of the infrapatellar fat pad (IPFP) of OA or cartilage defect patients by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), which could be used as biomarkers for early OA detection. A higher presence of phospholipids was found in OA patients compared with cartilage defect patients. In addition, a higher abundance of ether-linked phosphatidylethanolamines (PE O-s) containing arachidonic acid was specifically found in OA patients compared with cartilage defect patients. These lipids were mainly found in the connective tissue of the IPFP. Specific lipid species were associated to OA patients compared with cartilage defect patients. PE O-s have been suggested as possible biomarkers for OA. As these were found more abundantly in the connective tissue, the IPFP's intra-tissue heterogeneity might play an important role in biomarker discovery, implying that the amount of fibrous tissue is associated with OA.
Assuntos
Osteoartrite do Joelho , Humanos , Idoso , Osteoartrite do Joelho/diagnóstico por imagem , Osteoartrite do Joelho/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Tecido Adiposo/patologia , Biomarcadores , Biópsia , Cartilagem/patologia , Lipídeos , LasersRESUMO
MALDI MS imaging (MSI) is a powerful analytical tool for spatial peptide detection in heterogeneous tissues. Proper sample preparation is crucial to achieve high quality, reproducible measurements. Here we developed an optimized protocol for spatially resolved proteolytic peptide detection with MALDI time-of-flight MSI of fresh frozen prostate tissue sections. The parameters tested included four different tissue washes, four methods of protein denaturation, four methods of trypsin digestion (different trypsin densities, sprayers, and incubation times), and five matrix deposition methods (different sprayers, settings, and matrix concentrations). Evaluation criteria were the number of detected and excluded peaks, percentage of high mass peaks, signal-to-noise ratio, spatial localization, and average intensities of identified peptides, all of which were integrated into a weighted quality evaluation scoring system. Based on these scores, the optimized protocol included an ice-cold EtOH+H2 O wash, a 5 min heating step at 95°C, tryptic digestion incubated for 17h at 37°C and CHCA matrix deposited at a final amount of 1.8 µg/mm2 . Including a heat-induced protein denaturation step after tissue wash is a new methodological approach that could be useful also for other tissue types. This optimized protocol for spatial peptide detection using MALDI MSI facilitates future biomarker discovery in prostate cancer and may be useful in studies of other tissue types.