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Triclosan (TCS), an antimicrobial compound, is known to have potential endocrine-disruptive properties, but the underlying toxic mechanisms at the metabolic level are not well understood. Here, we applied metabolomics and lipidomics combined with mass spectrometry imaging (MSI) to unveil the mechanisms of the enhanced growth of MCF-7 breast cancer cell spheroids (CCS) exposed to TCS. To obtain a wide coverage of metabolites and lipids by using MSI, we used techniques of matrix-assisted laser desorption/ionization (MALDI) and MALDI coupled with laser-postionization. The results showed that TCS and TCS sulfate penetrated into the entire area at 0-3 h and both localized in the inner area at 6 h. After 24 h, a portion of two compounds was released from CCS. Omic data indicated that TCS exposure induced alterations via several pathways, including energy metabolism and biosynthesis of glycerophospholipids and glycerolipids. Further MSI data revealed that the enhancement of energy supply in the peripheral area and the increase of energy storage in the inner area might contribute to the enhanced growth of MCF-7 breast CCS exposed to TCS. This study highlights the importance of integrating metabolite distributions and metabolic profiles to reveal the novel mechanisms of TCS-triggered endocrine disrupting effects.
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Neoplasias da Mama , Triclosan , Humanos , Feminino , Triclosan/toxicidade , Lipidômica , Metabolômica/métodos , Metaboloma , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
The application of mass spectrometry imaging (MSI) to explore the responses of cancer cell spheroids (CCS) after treatment of exogenous molecules has attracted growing attention. Increasing studies have utilized MSI to image the two-dimensional distributions of exogenous and endogenous molecules in planar CCS sections. However, because CCS are volumetric and heterogenous, maintaining their three-dimensional (3D) information is essential for acquiring a better understanding of the tumor microenvironment and mechanisms of action of exogenous molecules. Here, an established method of 3D MSI was applied to distinguish the distributions of triclosan sulfate and endogenous lipids in three microregions of colon CCS with an enhanced growth induced by the treatment of triclosan, a common antimicrobial agent. The results of 3D MSI showed that triclosan sulfate gradually accumulated from the periphery to the entire structure of CCS and finally localized in the core region. Spatial lipidomics analysis revealed that the upregulated phosphatidylethanolamine (fold change (FD) = 1.26, p = 0.0021), phosphatidylinositol (FD = 1.17, p = 0.0180), and phosphatidylcholine (FD = 1.22, p = 0.0178) species mainly distributed in the outer proliferative region, while the upregulated sphingomyelin (FD = 1.18, p = 0.024) species tended to distribute in the inner necrotic region. Our results suggest that a competitive mechanism between inhibiting and promoting CCS growth might be responsible for the proliferation of CCS treated with triclosan.
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Neoplasias do Colo , Triclosan , Neoplasias do Colo/tratamento farmacológico , Humanos , Espectrometria de Massas/métodos , Fosfatidilcolinas , Fosfatidiletanolaminas , Fosfatidilinositóis , Esfingomielinas , Sulfatos , Triclosan/farmacologia , Microambiente TumoralRESUMO
Imaging of lipids of whole-body specimens in two-dimensional (2D) analysis provides a global picture of the lipid changes in lipid-disturbed diseases, enabling a better understanding of lipid functions and lipid-modulation processes in different organs. However, 2D imaging of a single cross section can hardly characterize the whole-body lipid alterations. In this work, a three-dimensional matrix-assisted laser desorption/ionization mass spectrometry imaging (3D MALDI-MSI) approach was developed for analysis of whole-body zebrafish, for the first time, and applied to identify altered lipids and map their spatial distributions by using a zebrafish model of Niemann-Pick disease type C1 (NPC1), a neurovisceral lipid storage disorder causing both neurodegenerative disorder and visceral organ damage. The constructed 3D fish model provided comprehensive information on the 3D distribution of lipids of interest and allowed direct correlations between these lipids and organs of the fish. Obtained results revealed that several sphingolipids and phospholipids showed significant alterations and exhibited different localization patterns in various organs such as the brain, spinal cord, intestines, and liver-spleen region in the npc1 gene mutant fish compared to those of the wild type. The whole-body 3D MALDI-MSI approach revealed unique lipid signatures for different NPC1-affected organs, which might offer insights into the link between the impaired lipid storage and subsequent clinical symptoms, such as neurodegeneration and hepatosplenomegaly.
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Doença de Niemann-Pick Tipo C , Peixe-Zebra , Animais , Imageamento Tridimensional , Doença de Niemann-Pick Tipo C/diagnóstico por imagem , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , EsfingolipídeosRESUMO
Triclosan (TCS) is a ubiquitous antimicrobial used in many daily consumer products. It has been reported to induce endocrine disrupting effects at low doses in mammals, disturbing sex hormone function and thyroid function. The hypothalamus plays a crucial role in the maintenance of neuroendocrine function and energy homeostasis. We speculated that the adverse effects of TCS might be related to the disturbance of metabolic processes in hypothalamus. The present study aimed at investigating the effects of TCS exposure on the protein and metabolite profiles in hypothalamus of mice. Male C57BL/6 mice were orally exposed to TCS at the dosage of 10 mg/kg/d for 13 weeks. The hypothalamus was isolated and processed for mass spectrometry (MS)-based proteomics and metabolomics analyses. The results showed that a 10.6% decrease (P = 0.066) in body weight gain was observed in the TCS exposure group compared with vehicle control group. Differential analysis defined 52 proteins and 57 metabolites that delineated TCS exposed mice from vehicle controls. Among the differential features, multiple proteins and metabolites were found to play vital roles in neuronal signaling and function. Bioinformatics analysis revealed that these differentially expressed proteins and metabolites were involved in four major biological processes, including glucose metabolism, purine metabolism, neurotransmitter release, and neural plasticity, suggesting the disturbance of homeostasis in energy metabolism, mitochondria function, neurotransmitter system, and neuronal function. Our results may provide insights into the neurotoxicity of TCS and extend our understanding of the biological effects induced by TCS exposure.
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Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Metabolômica , Proteômica , Triclosan/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Biologia Computacional , Relação Dose-Resposta a Droga , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Triclosan/administração & dosagem , Triclosan/químicaRESUMO
Multicellular tumor spheroids (MCTS) have gained increasing attention in cancer research because they may closely mimic some physiological characteristics of solid tumors. MCTS have been considered as a useful three-dimensional cell model for evaluating the effect of exogenous molecules on tumor progression. However, little is known about the metabolic response in MCTS after exposure to exogenous molecules. Herein, we applied metabolomics combined with MALDI-mass spectrometry imaging (MSI) to investigate the proliferation of three-dimensional MDA-MB-231 breast cancer cell spheroids treated with bisphenol S (BPS). MSI data revealed that BPS, a common environmental contaminant, penetrated MCTS in 5 min and gradually localized in the core of MCTS within 4 h. Metabolomic data demonstrated that BPS exposure induced significant changes in the levels of 28 metabolites that are involved in several pathways, including purine metabolism and the tricarboxylic acid cycle. The MSI results showed that three upregulated metabolites (ATP, ADP, and AMP) acting major roles in energy supply distributed in the proliferative zone of cell spheroids, further indicating the proliferative response of MDA-MB-231 cell spheroids caused by BPS exposure. One downregulated metabolite (xanthine) associated with reactive oxidative stress was found to localize toward the inner region of cell spheroids. These MSI results demonstrated that the increase of energy supply in the outer layer of cell spheroids might be responsible for BPS-induced proliferative response. Taken together, this integrated method might offer a more accurate and intuitive assessment for the effect of exogenous molecules on cancer progression.
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Compostos Benzidrílicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Poluentes Ambientais/farmacologia , Metaboloma/efeitos dos fármacos , Fenóis/farmacologia , Esferoides Celulares/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia Líquida , Humanos , Metabolômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Increasing studies have utilized mass spectrometry imaging (MSI) that is a label-free tool to investigate drug penetration and drug biotransformation in multicellular tumor spheroids (MCTS). Currently, the gelatin-assisted sectioning method is widely used to prepare frozen sections of MCTS for MSI. However, owing to the limited transparency of frozen gelatin, MCTS with diameters less than 500 µm that closely mimic solid tumors are difficult to be detected when cryosectioning. In order to identify the presence of MCTS, hematoxylin and eosin staining for frozen sections and dye pretreatment for MCTS were employed in previous works, which either increased the analytical time and cost in sample preparation or caused signal suppression in sample analysis. Herein, a new sectioning method was developed to prepare MCTS frozen sections. MCTS was coated with ice to ensure good visibility for small-size MCTS. The optimal cutting temperature compound was added around the ice block to assist the formation of frozen sections. A precast frozen mold was prepared to allow the acquisition of complete MCTS frozen sections. The developed method was applied to investigate lipid distribution in MCTS by using matrix-assisted laser desorption/ionization MSI. Compared to the gelatin-assisted sectioning method, our method did not cause signal suppression and analyte delocalization. Thus, this method provides an easy, universal, and innovative strategy to prepare MCTS frozen sections for further MSI analysis. Besides, we applied our method to investigate the penetration of bisphenol A in MCTS.
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Secções Congeladas , Gelo , Neoplasias/diagnóstico por imagem , Esferoides Celulares/química , Humanos , Espectrometria de Massas , Manejo de Espécimes , Células Tumorais CultivadasRESUMO
As a bisphenol A (BPA) alternative, bisphenol F (BPF) has been detected in various products, such as paper products, personal care products, and food. More importantly, the toxicity of BPF remains underexplored. We reported an integrated method to study the immunotoxic potentials and the underlying mechanisms of BPF on cell apoptosis, macrophage polarization, reactive oxygen species generation, expression and secretion of immune-related cytokines, and reprogramming of lipid signaling. More serious to BPA, BPF induced apoptosis in macrophages. The apoptosis was induced by activating both sphingomyelin-ceramide signaling pathway and oxidative stress, which included intrinsic (bax and caspase-9) and extrinsic apoptotic pathways (tumor necrosis factor receptor 1, caspase-8, and caspase-3). BPF exposure also induced the proinflammatory phenotype of the macrophage. This alternation was shown to be closely correlated with the modulation of biosynthesis and degradation of glycerophospholipids. This study demonstrated novel evidence that BPF as a substituent of BPA induced immunotoxic effects at environmentally relevant concentrations. We also showed that the reprogramming of lipidome plays a key role in the regulation of macrophage polarization and the induction of immunotoxicity of the BPA analogue.
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Compostos Benzidrílicos , Transdução de Sinais , Lipídeos , Macrófagos , Estresse Oxidativo , FenóisRESUMO
With the continuous exposure of environmental pollutants in organisms, determination of abundance variation and spatial distribution of lipids might expand our understanding of toxicological mechanisms occurring in the kidney. Herein, an integrated method involving mass spectrometry (MS)-based lipidomics and matrix-assisted laser desorption/ionization-MS imaging (MALDI-MSI) was developed for the study of nephrotoxicity in mice exposed to 10 and 100 µg bisphenol S (BPS)/kg body weight/day. The BPS exposure remarkable perturbed abundances of 91 potential markers that mainly involved in five metabolic pathways. We elucidated the lipids spatial heterogeneity by using morphological analysis, probabilistic latent semantic analysis, and coregistered multimodal three-dimensional (3D)-MSI. In morphological analysis, both 10 and 100 µg BPS induced significant nephrotoxicity to mice, including glomerular necrosis in renal cortex, cloudy swelling in renal medulla, and interstitial collapsing in renal pelvis. Significant differential signaling lipids such as sphingomyelin (SM) (d22:0/20:4), ceramide (Cer) (d18:2/24:1), and sphingosine (d18:0) related to inflammation were found to be up-regulated and colocalized in the renal cortex, medulla, and pelvis, respectively. Also, seven significant differential lipids, which are considered to be involved in membrane homeostasis and cellular function, were found to be colocalized in the renal cortex. The observed significant variations of morphology, lipid accumulation, and metabolism in the renal cortex implicated that lipids in the renal cortex were more sensitive to BPS exposure than those in the renal medulla and pelvis. Moreover, we reconstructed a 3D-MSI model of kidney and identified two heterogeneous-related substructures in the renal cortex and pelvis upon 100 µg BPS exposure. It might be used in novel specificity evaluation and early diagnosis for environmental pollutant-induced kidney diseases.
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Nefropatias/metabolismo , Lipídeos de Membrana/metabolismo , Metabolômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Feminino , Humanos , Nefropatias/induzido quimicamente , Camundongos Endogâmicos BALB C , Fenóis , Sulfonas , Distribuição TecidualRESUMO
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF MSI) is rapidly maturing as an innovative technique for spatial molecule ( m/ z > 1000 Da) profiling. However, direct identification of low-molecular-weight compounds ( m/ z < 600 Da) by MALDI-TOF MSI using conventional organic matrixes remains a challenge because of ionization suppression and serious matrix-related background interference. Furthermore, the heterogeneous cocrystallization that is inherent to organic matrixes can degrade spatial resolution in MSI. Herein, we developed a negative ion surface-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (SALDI-TOF MSI) protocol to detect bisphenol S (BPS) and map its spatial distribution in mouse tissues by applying nitrogen- and sulfur-co-doped carbon dots (N,S-co-doped CDs) as a new matrix through spraying. The SALDI-TOF MS and imaging parameters, such as matrix concentration, ionization mode, and matrix deposition, were optimized to improve imaging performance. In comparison to organic matrixes, the use of N,S-co-doped CDs in negative ion mode exhibited free matrix background interference, enhanced MS signal intensity, and provided high spatial resolution (acquired at â¼50 µm) in the analysis of BPS, which allowed sensitive detection of the target compound on the surfaces of tissue sections. Quantitative assessment was also made by spotting BPS standards directly onto the tissue surface, and a good correlation between the color change and BPS concentrations was found. The corresponding detection limit as low as the â¼pmol level for BPS was observed with the direct visualization from MS images. Furthermore, the feasibility of the proposed SALDI-TOF MSI method was extended for in situ identification of exogenous BPS in the different tissues of mouse involving liver, kidney, spleen, and heart for exposure and profiling its spatial localization at different administration times. In addition, the general applicability of the proposed method was also evaluated by SALDI-TOF MSI analysis of BPAF in tissues. These successful applications of SALDI-TOF MSI not only demonstrated its promising potential as an alternative to MALDI-TOF MSI in profiling small molecules in tissue sections but also provided tremendous insight into the assessment of BPS exposure.
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Carbono/química , Nitrogênio/química , Fenóis/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sulfonas/metabolismo , Enxofre/química , Animais , Camundongos , Camundongos Endogâmicos BALB C , Distribuição TecidualRESUMO
RATIONALE: As an important substitute of bisphenol A (BPA), bisphenol S (BPS) shows comparable estrogenic effects. BPS is now widely used in consumer products with widespread human exposure. In order to evaluate the health risk of BPS, it is essential to develop a rapid method for the determination of BPS and its metabolites as well as its biotransformation capacity in vivo. METHODS: Two major BPS phase II metabolites, BPS glucuronide (BPS-G) and BPS sulfate (BPS-S), were synthesized and used as standards for the development of a ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS) method. Phase II metabolism of BPS in imprinting control region (ICR) female mice after oral administration with different dosages (10, 100, 1000 µg/kg body weight) was investigated. RESULTS: Urinary elimination was the main excretion route for BPS, with the total recovery ranging from 52.8% to 78.1%. In urine, BPS-G was identified as the predominant metabolite, and the maximum concentrations of BPS-G and BPS-S were obtained at 6 h after the oral administration upon the adjustment by creatinine. BPS was the major compound existed in feces. Only trace amounts of BPS and its metabolites were detected in digestive and excretory related tissues (<1%). CONCLUSIONS: The distribution and metabolic pathway of BPS in mice were assessed. More than 50% of BPS was excreted through phase II metabolism. Due to the biological inactivity of BPS-G and BPS-S, rapid metabolism of BPS to BPS-G and BPS-S may result in reduced toxicity of BPS in vivo.
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Gold nanoparticles (AuNPs) have been frequently utilized for the construction of diverse colorimetric biosensors. Normally, AuNPs with sharp edges could have better sensitivity. However, the poor monodipersity of AuNPs with sharp edges seriously confines their utility for colorimetric biosensing. Herein, we demonstrate the utility of highly uniform gold nanobipyramids (Au NBPs) for ultrasensitive colorimetric detection of H5N1 virus. The proposed method is based on the fact that alkaline phosphatase (ALP) could catalyze the decomposition of 4-aminophenyl phosphate (4-APP) to generate 4-aminophenol (4-AP), which would then reduce silver nitrate to metal silver and then deposited on Au NBPs. The metal silver shell coated on the Au NBPs changed the refractive index of gold and thus resulted in a blue shift of longitudinal localized surface plasmon resonance (LSPR) and accompanied a vivid color change. This method exhibited a higher sensitivity than that of other Au NPs such as gold nanorods due to the high-index-faceted on the tips of the Au NBPs. This method was used to detect the activity of ALP. It exhibited a linear range of 0.1-5 mU/mL with a limit of detection (LOD) of 0.086 mU/mL. Finally, the proposed method was used in immunoassay to detect H5N1 virus. The results showed that the corresponding linear range for the detection of H5N1 virus antigen was 0.001-2.5 ng/mL, and the LOD was determined to be 1 pg/mL, which is more sensitive than those in most of the colorimetric biosensors reported previously.
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Colorimetria , Ouro/química , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Nanopartículas Metálicas/química , Fosfatase Alcalina/química , Fosfatase Alcalina/metabolismo , Aminofenóis/química , Humanos , Imunoensaio , Limite de Detecção , Ressonância de Plasmônio de SuperfícieRESUMO
Prenatal exposure to benzotriazoles and benzothiazoles (collectively as BTs) was associated with pregnancy complications. Identifying the metabolites associated with prenatal BTs exposure may help elucidate the mechanism and characterize the exposure risk. In this prospective study of 158 pregnant women from Wuhan, China, urinary BTs were repeatedly measured across three trimesters to provide an accurate estimation of exposure during pregnancy. We conducted high-throughput targeted metabolomics with great coverage and high accuracy to characterize the urinary metabolic profile in late pregnancy. We first identified the perturbed metabolites of cocktail BTs exposure and then pinned down to the pairwise associations between individual BTs and the identified metabolites. A total of 44 metabolites were identified as perturbed biomarkers of cocktail BTs exposure based on the variable influence on projection (VIP > 1.2) score. Further pairwise associations analysis showed positive association of BTs with oxidative stress related biomarkers and negative association of BTs with neuronal function metabolites. The shared metabolic signatures among BTs in the co-occurrence network of pairwise association analysis may partially be attributed to the correlation among cocktail BTs exposure. The findings provide the potential mechanisms of BTs-associated pregnancy complications and offer insight into the health implications for prenatal BTs exposure. Furthermore, the framework we employed, which integrates both cocktail exposure and individual exposure, may illuminate future epidemiological research that seeks to incorporate exposure to mixtures and omics scale data.
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Benzotiazóis , Biomarcadores , Exposição Materna , Triazóis , Feminino , Gravidez , Triazóis/toxicidade , Humanos , Adulto , Biomarcadores/urina , Biomarcadores/metabolismo , Estudos Prospectivos , China , Metabolômica , Metaboloma/efeitos dos fármacosRESUMO
INTRODUCTION: Bacterial living states and the distribution of microbial colony signaling molecules are widely studied using mass spectrometry imaging (MSI). However, current approaches often treat 3D colonies as flat 2D disks, inadvertently omitting valuable details. The challenge of achieving 3D MSI in biofilms persists due to the unique properties of microbial samples. OBJECTIVES: The study aimed to develop a new biofilm sample preparation method that can realize high-resolution 3D MSI of bacterial colonies to reveal the spatial organization of bacterial colonies. METHODS: This article introduces the moisture-assisted cryo-section (MACS) method, enabling embedding-free sectioning parallel to the growth plane. The MACS method secures intact sections by controlling ambient humidity and slice thickness, preventing molecular delocalization. RESULTS: Combined with matrix-assisted laser desorption ionization mass spectrometry (MALDI)-MSI, the MACS method provides high-resolution insights into endogenic and exogenous molecule distributions in Pseudomonas aeruginosa (P. aeruginosa) biofilms, including isomeric pairs. Moreover, analyzed colonies are revived into 3D models, vividly depicting molecular distribution from inner to outer layers. Additionally, we investigated metabolite spatiotemporal dynamics in multiple colonies, observing changes over time and distinct patterns in single versus merged colonies. These findings shed light on the repel-merge process for multi-colony formation. Furthermore, our study monitored chemical responses inside biofilms after antibiotic treatment, showing increased antibiotic levels in the outer biofilm layer over time while maintaining low levels in the inner region. Moreover, the MACS method demonstrated its universality and applicability to other bacterial strains. CONCLUSION: These results unveil complex cell activities within biofilm colonies, offering insights into microbe communities. The MACS method is universally applicable to loosely packed microorganism colonies, overcoming the limitations of previously reported MSI methods. It has great potential for studying bacterial-infected cancer tissues and artificial organs, making it a valuable tool in microbiological research.
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Seed germination is a vital process in plant development involving dynamic biochemical transformations such as lipid metabolism. However, the spatial distribution and dynamic changes of lipids in different seed compartments during germination are poorly understood. In this study, we employed liquid chromatography/mass spectrometry (LC/MS)-based lipidomics and MALDI mass spectrometry imaging (MSI) to investigate lipid changes occurring in the cotyledon and plumule of mung bean seeds during germination. Lipidomic data revealed that the germination process reduced the levels of many glycerolipids (e.g., triglyceride) and phosphatidylglycerols (e.g., phosphatidylcholine) while increased the levels of lysophospholipids (e.g., lysophosphatidylcholine) in both the cotyledon and plumule. Sphingolipids (e.g., sphingomyelin) displayed altered levels solely in the plumule. Sterol levels increased in the cotyledon but decreased in the plumule. Further imaging results revealed that MALDI-MSI could serve as a supplement and validate LC-MS data. These findings enhance our understanding of the metabolic processes underlying seedling development, with potential implications for crop improvement and seed quality control.
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Fabaceae , Vigna , Cotilédone/metabolismo , Germinação , Lipidômica , Sementes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triglicerídeos/metabolismoRESUMO
Lipids and metabolites are small biological molecules that act major roles in cellular functions. Multicellular tumor spheroids (MCTS) are a highly beneficial three-dimensional cellular model for cancer research due to their ability to imitate numerous characteristics of tumor tissues. Increasing studies have performed spatial lipidomics and metabolomics in MCTS using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). However, these approaches often lack the sensitivity and specificity to offer a comprehensive characterization of lipids and metabolites within MCTS. In this study, we addressed this challenge by utilizing MALDI combined with laser-induced postionization (MALDI-2) and trapped ion mobility spectrometry (TIMS) imaging in H295R adrenocortical MCTS. Our results showed that MALDI-2 could detect more lipids and metabolites in MCTS than the traditional MALDI. TIMS data revealed a successful separation of many isomeric and isobaric ions of lipids and metabolites with different locations (e.g., proliferative region and necrotic region) within MCTS, suggesting an enhanced peak capacity for spatial lipidomics and metabolomics. To further identify these isomeric and isobaric ions, we performed MS/MS imaging experiments to compare the differences in signal intensities and spatial distributions of product ions. Our data highlight the strong potential of MALDI-2 and TIMS imaging for analyzing lipids and metabolites in MCTS, which may serve as valuable tools for numerous fields of biological and medical research.
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Lipidômica , Neoplasias , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Lipídeos/química , Espectrometria de Massas em Tandem , Metabolômica/métodos , Íons/químicaRESUMO
The flexible and controlled synthesis of metal-organic framework (MOF)-derived hybrid nanostructures is of great significance in fine tuning of their enrichment performance in large-scale and in-depth phosphoproteome analysis. Herein, a magnetic guanidyl-functionalized MOF hybrid coating with multiaffinity sites, denoted as Fe3O4@G-ZIF-8, was fast fabricated via a one-pot epitaxial growth strategy for the first time and applied for selective and highly efficient enrichment of global phosphopeptides. The intrinsic unsaturated metal sites of ZIF-8 endow the surface-mounted MOF coatings with immobilized metal ion affinity chromatography interaction with multiphosphorylated peptides. The oriented anchoring of bifunctional guanidineacetic acid on the magnetic MOF nanospheres provides additional affinity sites (guanidyl groups) for specific recognition of phosphopeptides by "salt bridge" interaction, as well as active site carboxyl groups for the coordination with the metal ions. The as-prepared Fe3O4@G-ZIF-8 exhibits large surface area (382.5 m2 g-1), good superparamagnetic property (41.6 emu g-1) and stability, and size-exclusion effect (1.73 nm), which can serve as a specific adsorbent for global phosphopeptide analysis with satisfactory selectivity, great detection sensitivity (1 fmol), and rapid magnetic separation. Moreover, the successful application of Fe3O4@G-ZIF-8 for selective capture of both multi- and mono-phosphopeptides from human saliva and serum demonstrated the great potential of magnetic surface-mounted MOF coatings in effective identification of low-abundance phosphopeptides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry from complicated biological matrices.
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Estruturas Metalorgânicas , Fosfopeptídeos , Cromatografia de Afinidade/métodos , Humanos , Íons/química , Fenômenos Magnéticos , Magnetismo , Estruturas Metalorgânicas/química , Fosfopeptídeos/análiseRESUMO
Protein glycosylation is a ubiquitous and important biological process involved in various molecular functions and biological pathways. It also yields important biomarkers for clinical diagnoses. However, glycopeptide analysis is challenging due to low abundance, low ionization efficiency, and glycan heterogeneity. In the present study, a method based on hydrophilic interaction liquid chromatography (HILIC) was developed for the selective enrichment of glycopeptides using a novel metal-organic framework (MOF) nanocomposite (AuGC/ZIF-8). Dual functionalization with glutathione and cysteine has resulted in an ultra-hydrophilic MOF, with synergistic effects and lower steric hindrance, providing more affinity sites for the glycopeptide enrichment. Horseradish peroxidase (HRP) was used as a model glycoprotein, and AuGC/ZIF-8 was used to enrich glycopeptides prior to analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). AuGC/ZIF-8 displayed outstanding performance at enriching HRP glycopeptides, with high enrichment capacity (250 µg/mg), high selectivity in mixtures containing bovine serum albumin (BSA) (HRP-BSA (1â¶200, mass ratio)), and high sensitivity at very low content (0.3 ng/µL). Thus this MOF holds promise for in-depth, comprehensive glycoproteomic and related analysis.
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Glicopeptídeos/química , Estruturas Metalorgânicas , Nanocompostos , Cromatografia Líquida , Peroxidase do Rábano Silvestre , Interações Hidrofóbicas e Hidrofílicas , Estruturas Metalorgânicas/química , Soroalbumina Bovina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Three-dimensional (3D) multicellular tumor spheroids (MCTS) that mimic the complex tumor microenvironment provide a good platform for in vitro study of drug and endogenous metabolites. Hydroxychloroquine (HCQ) has shown anti-tumor activity in a variety of tumor models. However, the effect of the drug on the alteration of lipid metabolism spatial composition and distribution in the MCTS model is not clear. Herein, we utilized matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) in the analysis of A549 lung cancer multicellular spheroids to investigate the in situ spatial distribution of HCQ and its effect on lipid metabolism. We have successfully observed the spatial variations of HCQ in the inner region of the spheroid at different drug-treated time points. The MSI results also demonstrated that HCQ treatment altered the spatial composition of lipids in the inner and outer regions of treated spheroids. Furthermore, the lipidomic results showed that the identified phosphatidylcholines (PC), lysophosphatidylcholines (LPC), phosphatidylethanolamines (PE), lysophosphatidylethanolamines (LPE), phosphatidylinositols (PI), ceramides (Cer), glucosylceramides (CerG), and diglycerides (DG) were significantly up-regulated, and phosphatidylglycerol (PG) and triglycerides (TG) were remarkable down-regulated. MSI method combined with LC-MS/MS profiling of endogenous metabolites can obtain more detailed information about how spheroids respond to drug and spatial distribution information, thus fostering a better understanding of the relationship between drug-altered lipid metabolism and cancer microenvironment.
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Neoplasias Pulmonares , Esferoides Celulares , Cromatografia Líquida , Humanos , Hidroxicloroquina/farmacologia , Fosfatidilcolinas , Espectrometria de Massas em Tandem , Microambiente TumoralRESUMO
Gestational exposure to PM2.5 is associated with adverse postnatal outcomes. PM2.5 can enter alveoli by using intratracheal instillation, even penetrate through lung cells into the blood circulation. Subsequently, they are transferred across the placenta and fetal blood brain barrier, causing the adverse birth outcomes of offspring. This study demonstrated that the gestational exposure resulted in cognitive and emotional disorders in female offspring although the offspring were not exposed to PM2.5. Placental metabolic pathways modulated fetal brain development and played a pivotal role for maternal-placental-fetal interactions in the fetal programming of adult behavioral and mental disorders. Samples of fetus, offspring hippocampus and placenta from the mice exposed to PM2.5 were investigated using a comprehensive approach including mass spectrometry-based lipidomics and three-dimensional imaging. The exposure induced the neuro-degeneration in hippocampus, impairment of placental cytoarchitecture, and reprogramming of lipidome, which might affect the modulation of maternal-fetal cross-talk and result in the behavior disorders of offspring. The variation of spatial distribution of lipids was profoundly affected in dorsal pallium and hippocampal formation regions of fetal brain, offspring hippocampus, as well as labyrinth and junctional zones of placenta. The abundance alteration of lipid markers associated with neurodegenerative diseases was validated in transgenic mouse model with Alzheimer's disease and human cerebrospinal fluid from patients with Parkinson's disease. The finding could help with the selection of more suitable heterogeneous-related substructures targeting PM2.5 exposure and the exploration of PM2.5-induced toxicological effects on neurodegenerative diseases.
RESUMO
Previous in vivo exposure studies focused mainly on nuclear receptors involved in hepatotoxicity of triclosan (TCS). As liver plays a vital role in metabolic processes, dysregulations in lipid metabolism have been identified as potential drivers of pathogenesis. Investigation of changes in lipid metabolism might widen our understanding of toxicological effects as well as the underlying mechanism occurring in the liver. In this study, we comprehensively assessed the effect of TCS exposure on hepatic lipid metabolism in mice. Our results showed that TCS induced significant changes in hepatic free fatty acid pool by upregulation of fatty acid uptake and de novo fatty acid synthesis. Besides, hepatic levels of lipids, including acyl carnitine (AcCa), ceramide (Cer), triacylglycerols (TG), phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE) were also increased, together with upreguation of genes associated to TG synthesis, fatty acid oxidation and inflammation in TCS exposure group. These changes in lipid homeostasis could contribute to membrane instability, lipid accumulation, oxidative stress and inflammation. Our results suggested that TCS exposure could induce hepatic lipid metabolism disorders in mice, which would further contribute to the liver damage effects of TCS.