Spatiotemporal pharmacometabolomics based on ambient mass spectrometry imaging to evaluate the metabolism and hepatotoxicity of amiodarone in HepG2 spheroids

Three-dimensional(3D)cell spheroid models combined with mass spectrometry imaging(MSI)enables innovative investigation of in vivo-like biological processes under different physiological and pathological conditions.Herein,airflow-assisted desorption electrospray ionization-MSI(AFADESI-MSI)was coupled with 3D HepG2 spheroids to assess the metabolism and hepatotoxicity of amiodarone(AMI).High-coverage imaging of>1100 endogenous metabolites in hepatocyte spheroids was achieved using AFADESI-MSI.Following AMI treatment at different times,15 metabolites of AMI involved in Ndesethylation,hydroxylation,deiodination,and desaturation metabolic reactions were identified,and according to their spatiotemporal dynamics features,the metabolic pathways of AMI were proposed.Subsequently,the temporal and spatial changes in metabolic disturbance within spheroids caused by drug exposure were obtained via metabolomic analysis.The main dysregulated metabolic pathways included arachidonic acid and glycerophospholipid metabolism,providing considerable evidence for the mechanism of AMI hepatotoxicity.In addition,a biomarker group of eight fatty acids was selected that provided improved indication of cell viability and could characterize the hepatotoxicity of AMI.The combination of AFADESI-MSI and HepG2 spheroids can simultaneously obtain spatiotemporal information for drugs,drug metabolites,and endogenous metabolites after AMI treatment,providing an effective tool for in vitro drug hepatotoxicity evaluation.

Using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to analyze differentially expressed brain polypeptides in scrapie strain 22L-infected BALB/c mice

Differentialiy expressed polypeptides in the brain of a BALB/c mouse model infected with scrapie strain 22L were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results showed that 21 peptides were down-regulated, with peptides of mass-to-charge ratio 758.772 5 and mass-to-charge ratio 5 432.206 9, demonstrating the most significant decreases. These finding suggest that these peptides are candidate biomarkers and may play an important role in the pathogenesis of prion disease.

Analysis of the Spectral Resolving Power in Tomographic Imaging Spectrometry

From the point of view of design requirements, influence of the width of the output image of an imaging subsystem in a tomographic imaging spectrometer, namely width of the slit, the grating and the size of the CCD pixel are analyzed. For the tomographic imaging spectrometry, if the amplification ratio of the imaging subsystem is not high enough to make the whole object to be compressed within the slit, then either the slit width should be increased or the slit width kept unchanged but scanned to receive information of the object. While the width-increase method reduces the spectral resolving power and the SNR; the scanning method reduces the SNR. Analysis of the two cases and computer simulation results are given.

Integrated mass spectrometry imaging reveals spatial-metabolic alteration in diabetic cardiomyopathy and the intervention effects of ferulic acid

Diabetic cardiomyopathy(DCM)is a metabolic disease and a leading cause of heart failure among people with diabetes.Mass spectrometry imaging(MSI)is a versatile technique capable of combining the molecular specificity of mass spectrometry(MS)with the spatial information of imaging.In this study,we used MSI to visualize metabolites in the rat heart with high spatial resolution and sensitivity.We optimized the air flow-assisted desorption electrospray ionization(AFADESI)-MSI platform to detect a wide range of metabolites,and then used matrix-assisted laser desorption ionization(MALDI)-MSI for increasing metabolic coverage and improving localization resolution.AFADESI-MSI detected 214 and 149 metabolites in positive and negative analyses of rat heart sections,respectively,while MALDI-MSI detected 61 metabolites in negative analysis.Our study revealed the heterogenous metabolic profile of the heart in a DCM model,with over 105 region-specific changes in the levels of a wide range of metabolite classes,including carbohydrates,amino acids,nucleotides,and their derivatives,fatty acids,glycerol phospholipids,carnitines,and metal ions.The repeated oral administration of ferulic acid during 20 weeks significantly improved most of the metabolic disorders in the DCM model.Our findings provide novel insights into the molecular mechanisms underlying DCM and the potential of ferulic acid as a therapeutic agent for treating this condition.

Spatiotemporal visualization of the synthesis and accumulation of rotenone in Derris elliptica roots using mass spectrometry imaging

The discovery of novel botanical pesticides as a preferred alternative to synthetic pesticides is regarded as an environmentally friendly strategy,yet it remains a great challenge due to limited insights into the synthesis and accumulation of active ingredient intermediates.Herein,we demonstrate the use of gold nanoparticle(AuNP)-assisted laser desorption/ionization mass spectrometry imaging(LDI-MSI)for the tissue-specific distribution and spatiotemporal accumulation effect of rotenone and active ingredient intermediates in its biosynthetic pathway within the roots of Derris elliptica.The MSI results revealed that rotenone was mainly concentrated in the epidermis,cortex,and xylem in the first two years and began to accumulate in the phloem since the 3rd year.Meanwhile,the rotenone contents increased in the epidermis,cortex,and xylem in the 4th year and finally decreased in the 5th year.The ultra-performance liquid chromatography(UPLC)results revealed that there was a significant difference in rotenone content between the root and non-root bark(p<0.05)except for the 1st year.It reached its maximum value at 9.71%in the root bark in the 4th year and 7.98%in the non-root bark in the 1st year,which was in accordance with the MSI results.Additionally,the active ingredient intermediates in the Hydroxylation/Methylation and Rotenoid Phases of the biosynthetic pathway of rotenone were concentrated in the root region.Taken together,the collective results provide the scientific guidance for the scientific cultivation of D.elliptica and the efficient extraction of botanical pesticides.

Spatially resolved metabolomics visualizes heterogeneous distribution of metabolites in lung tissue and the anti-pulmonary fibrosis effect of Prismatomeris connate extract

Pulmonary fibrosis (PF) is a chronic progressive end-stage lung disease. However, the mechanisms underlying the progression of this disease remain elusive. Presently, clinically employed drugs are scarce for the treatment of PF. Hence, there is an urgent need for developing novel drugs to address such diseases. Our study found for the first time that a natural source of Prismatomeris connata Y. Z. Ruan (Huang Gen, HG) ethyl acetate extract (HG-2) had a significant anti-PF effect by inhibiting the expression of the transforming growth factor beta 1/suppressor of mothers against decapentaplegic (TGF-β1/Smad) pathway. Network pharmacological analysis suggested that HG-2 had effects on tyrosine kinase phosphorylation, cellular response to reactive oxygen species, and extracellular matrix (ECM) disassembly. Moreover, mass spectrometry imaging (MSI) was used to visualize the heterogeneous distribution of endogenous metabolites in lung tissue and reveal the anti-PF metabolic mechanism of HG-2, which was related to arginine biosynthesis and alanine, asparate and glutamate metabolism, the downregulation of arachidonic acid metabolism, and the upregulation of glycerophospholipid metabolism. In conclusion, we elaborated on the relationship between metabolite distribution and the progression of PF, constructed the regulatory metabolic network of HG-2, and discovered the multi-target therapeutic effect of HG-2, which might be conducive to the development of new drugs for PF.

Spatial metabolomics reveal metabolic alternations in the injured mice kidneys induced by triclocarban treatment

Triclocarban(TCC)is a common antimicrobial agent that has been widely used in medical care.Given the close association between TCC treatment and metabolic disorders,we assessed whether long-term treatment to TCC at a human-relevant concentration could induce nephrotoxicity by disrupting the metabolic levels in a mouse model.Matrix-assisted laser desorption/ionization mass spectrometry imaging(MALDI-MSI)was applied to investigate the alterations in the spatial distributions and abundances of TCC,endogenous and exogenous metabolites in the kidney after TCC treatment.The results showed that TCC treatment induced the changes in the organ weight,organ coefficient and histopathology of the mouse kidney.MSI data revealed that TCC accumulated in all regions of the kidney,while its five metabolites mainly distributed in the cortex regions.The abundances of 79 biomolecules associated with pathways of leukotriene E4 metabolism,biosynthesis and degradation of glycerophospholipids and glycerolipids,ceramide-to-sphingomyelin signaling were significantly altered in the kidney after TCC treatment.These biomolecules showed distinctive distributions in the kidney and displayed a favorable spatial correlation with the pathological damage.This work offers new insights into the related mechanisms of TCC-induced nephrotocicity and exhibits the potential of MALDI-MSI-based spatial metabolomics as a promising approach for the risk assessment of agents in medical care.

质谱成像方法及其在临床研究中的应用

Imaging mass spectrometry (IMS) methodology for biological tissue component distribution map using MALDI-TOF MS and MALDI-TOF/TOF MS was established.Peak density distribution was probed to be quite useful for MS image classification.More than 40 000 spectra from 200 tissue sections were acquired and reproducibility between various of species groups was great than 80%.Tens of differentiately expressed components were detected by t-test (P<0.01).Classification modeling was created based on the differentiate components,blind species were analyzed for model validation,accuracy was above 90%.

Dual mass spectrometry imaging and spatial metabolomics to investigate the metabolism and nephrotoxicity of nitidine chloride

Evaluating toxicity and decoding the underlying mechanisms of active compounds are crucial for drug development.In this study,we present an innovative,integrated approach that combines air flowassisted desorption electrospray ionization mass spectrometry imaging(AFADESI-MSI),time-of-flight secondary ion mass spectrometry(ToF-SIMS),and spatial metabolomics to comprehensively investigate the nephrotoxicity and underlying mechanisms of nitidine chloride(NC),a promising anti-tumor drug candidate.Our quantitive AFADESI-MSI analysis unveiled the region specific of accumulation of NC in the kidney,particularly within the inner cortex(IC)region,following single and repeated dose of NC.High spatial resolution ToF-SIMS analysis further allowed us to precisely map the localization of NC within the renal tubule.Employing spatial metabolomics based on AFADESI-MSI,we identified over 70 discriminating endogenous metabolites associated with chronic NC exposure.These findings suggest the renal tubule as the primary target of NC toxicity and implicate renal transporters(organic cation transporters,multidrug and toxin extrusion,and organic cation transporter 2(OCT2)),metabolic enzymes(protein arginine N-methyltransferase(PRMT)and nitric oxide synthase),mitochondria,oxidative stress,and inflammation in NC-induced nephrotoxicity.This study offers novel insights into NC-induced renal damage,representing a crucial step towards devising strategies to mitigate renal damage caused by this compound.

Discovering metabolic vulnerability using spatially resolved metabolomics for antitumor small molecule-drug conjugates development as a precise cancer therapy strategy

Against tumor-dependent metabolic vulnerability is an attractive strategy for tumor-targeted therapy.However,metabolic inhibitors are limited by the drug resistance of cancerous cells due to their metabolic plasticity and heterogeneity.Herein,choline metabolism was discovered by spatially resolved metabolomics analysis as metabolic vulnerability which is highly active in different cancer types,and a choline-modified strategy for small molecule-drug conjugates(SMDCs)design was developed to fool tumor cells into indiscriminately taking in choline-modified chemotherapy drugs for targeted cancer therapy,instead of directly inhibiting choline metabolism.As a proof-of-concept,choline-modified SMDCs were designed,screened,and investigated for their druggability in vitro and in vivo.This strategy improved tumor targeting,preserved tumor inhibition and reduced toxicity of paclitaxel,through targeted drug delivery to tumor by highly expressed choline transporters,and site-specific release by carboxylesterase.This study expands the strategy of targeting metabolic vulnerability and provides new ideas of developing SMDCs for precise cancer therapy.

Expansion Strategy-Driven Micron-Level Resolution Mass Spectrometry Imaging of Lipids in Mouse Brain Tissue

A novel method for enhanced resolution,termed expansion mass spectrometry imaging,has been developed for lipid mass spectrometry imaging,utilizing existing commercially available mass spectrometers without necessitating modifications.This approach involves embedding tissue sections in a swellable polyelectrolyte gel,with the target biomolecules indirectly anchored to the gel network.By employing matrix-assisted laser desorption ionization mass spectrometry imaging,the method has realized an enhanced spatial resolution that surpasses the conventional resolution limits of commercial instruments by approximately 4.5 fold.This enhancement permits the detailed visualization of intricate structures within the mouse brain at a subcellular level,with a lateral resolution nearing 1μm.As a physical technique for achieving resolution beyond standard capabilities,this readily adaptable approach presents a powerful tool for high-definition imaging in biological research.

Mass Spectrometric Imaging of Gold Nanolayer Coated Latent Fingermarks: Deciphering Overlapping Features by Statistical Analysis

Overlapping latent fingermarks constitute a serious challenge to database related recognition and matching algorithms in biometry, forensic and crime scene investigations. Mass spectrometry imaging (MSI) is a powerful tool for deciphering and analyzing overlapping fingermarks based on the individual chemical information of each deposit. Fingermark MSI in practice still requires a subjective judgment of an MSI expert, such that rapid analysis, automation, standardization, and a quantitative evaluation of the complete detection and separation process of overlapped fingermarks from MSI data sets is the ultimate goal and will be necessary to become an accepted process in criminal investigations and law enforcement. Here we investigated the feasibility and efficiency of different statistical approaches for the separation of overlapped latent fingermarks based on MSI data. Entropy analysis of generated m/z-images was used to evaluate the results obtained from the statistical analysis. Furthermore, we demonstrate and discuss the opportunity to reconstitute and separate overlapping fingermarks by discrete scanning at selected x,y-positions defined from a previous image analysis using a more simple schema based on visible and therefore optical distinguishable overlapped ink-based fingermarks. The overlapped latent fingermarks were developed by rapid gold sputter coating and analyzed by laser based MSI, without (organic) matrix preparation. Latent finger marks can be transferred from the substrate/surface with and conserved on a soft gold sputtered soft membrane at low temperatures.

MALDI coupled with laser-postionization and trapped ion mobility spectrometry contribute to the enhanced detection of lipids in cancer cell spheroids

Cancer cell spheroids(CCS) are a valuable three-dimensional cell model in cancer studies because they could replicate numerous characteristics of solid tumors. Increasing researches have used matrix-assisted laser desorption/ionization mass spectrometry imaging(MALDI-MSI) to investigate the spatial distribution of endogenous compounds(e.g., lipids) in CCS. However, only limited lipid species can be detected owing to a low ion yield by using MALDI. Besides, it is still challenging to fully characterize the structural diversity of lipids due to the existence of isomeric/isobaric species. Here, we carried out the initial application of MALDI coupled with laser-postionization(MALDI-2) and trapped ion mobility spectrometry(TIMS) imaging in HCT116 colon CCS to address these challenges. We demonstrated that MALDI-2 is capable of detecting more number and classes of lipids in HCT116 colon CCS with higher signal intensities than MALDI. TIMS could successfully separate numerous isobaric/isomeric species of lipids in CCS. Interestingly, we found that some isomeric/isobaric species have totally different spatial distributions in colon CCS. Further MS/MS imaging analysis was employed to determine the compositions of fatty acid chains for isomeric species by examining disparities in signal intensities and spatial distributions of product ions. This work stresses the robust ability of TIMS and MALDI-2 imaging in analyzing endogenous lipids in CCS, which could potentially become powerful tools for future cancer studies.

An Evaluation of Spaceborne Imaging Spectrometry for Estimation of Forest Canopy Nitrogen Concentration in a Subtropical Conifer Plantation of Southern China

Canopy foliar Nitrogen Concentration （CNC） is one of the most important parameters influencing vegetation productivity in forest ecosystems. In this study, we explored the potential of imaging spectrometry （hyperspectral） remote sensing of CNC in conifer plantations in China’s subtropical red soil hilly region. Our analysis included data from 57 field plots scattered across two transects covered by Hyperion images. Single regression and partial least squares regression （PLSR） were used to explore the relationships between CNC and hyperspectral data. The correlations between CNC and nearinfrared relfectance （NIR） were consistent in three data subsets （subsets A-C）. For all subsets, CNC was signiifcantly positively correlated with NIR in the two transects （R2=0.29, 0.33 and 0.36, P＆lt;0.05 or P＆lt;0.01, respectively）. It suggested that the NIR-CNC relationship exist despite a weak one, and the relationship may be weakened by the single canopy structure. Besides, we also applied a shortwave infrared （SWIR） index - Normalized Difference Nitrogen Index （NDNI） to estimate CNC variation. NDNI presented a signiifcant positive correlation with CNC in different subsets, but like NIR, it was also with low coefifcient of determination （R2=0.38, 0.20 and 0.17, P＆lt;0.01, respectively）. Also, the correlations between CNC and the entire spectrum reflectance （or its derivative and logarithmic transformation） by PLSR owned different signiifcance in various subsets. We did not ifnd the very robust relationship like previous literatures, so the data we used were checked again. The paired T-test was applied to estimate the inlfuence of inter-annual variability of FNC on the relationships between CNC and Hyperion data. The inter-annual mismatch between period of ifeldwork and Hyperion acquisition had no inlfuence on the correlations of CNC-Hyperion data. Meanwhile, we pointed out that the lack of the canopy structure variation in conifer plantation area may lead to these weak relationships.

In situ metabolomics in nephrotoxicity of aristolochic acids based on air flow-assisted desorption electrospray ionization mass spectrometry imaging

Understanding of the nephrotoxicity induced by drug candidates is vital to drug discovery and development.Herein,an in situ metabolomics method based on air flow-assisted desorption electrospray ionization mass spectrometry imaging(AFADESI-MSI)was established for direct analysis of metabolites in renal tissue sections.This method was subsequently applied to investigate spatially resolved metabolic profile changes in rat kidney after the administration of aristolochic acid I,a known nephrotoxic drug,aimed to discover metabolites associated with nephrotoxicity.As a result,38 metabolites related to the arginine-creatinine metabolic pathway,the urea cycle,the serine synthesis pathway,metabolism of lipids,choline,histamine,lysine,and adenosine triphosphate were significantly changed in the group treated with aristolochic acid I.These metabolites exhibited a unique distribution in rat kidney and a good spatial match with histopathological renal lesions.This study provides new insights into the mechanisms underlying aristolochic acids nephrotoxicity and demonstrates that AFADESI-MSI-based in situ metabolomics is a promising technique for investigation of the molecular mechanism of drug toxicity.

Spatial-resolved metabolomics reveals tissue-specific metabolic reprogramming in diabetic nephropathy by using mass spectrometry imaging

Detailed knowledge on tissue-specific metabolic reprogramming in diabetic nephropathy(DN)is vital for more accurate understanding the molecular pathological signature and developing novel therapeutic strategies.In the present study,a spatial-resolved metabolomics approach based on air flowassisted desorption electrospray ionization(AFADESI)and matrix-assisted laser desorption ionization(MALDI)integrated mass spectrometry imaging(MSI)was proposed to investigate tissue-specific metabolic alterations in the kidneys of high-fat diet-fed and streptozotocin(STZ)-treated DN rats and the therapeutic effect of astragalosideⅣ,a potential anti-diabetic drug,against DN.As a result,a wide range of functional metabolites including sugars,amino acids,nucleotides and their derivatives,fatty acids,phospholipids,sphingolipids,glycerides,carnitine and its derivatives,vitamins,peptides,and metal ions associated with DN were identified and their unique distribution patterns in the rat kidney were visualized with high chemical specificity and high spatial resolution.These region-specific metabolic disturbances were ameliorated by repeated oral administration of astragaloside Ⅳ(100 mg/kg)for 12 weeks.This study provided more comprehensive and detailed information about the tissue-specific metabolic reprogramming and molecular pathological signature in the kidney of diabetic rats.These findings highlighted the promising potential of AFADESI and MALDI integrated MSI based metabolomics approach for application in metabolic kidney diseases.

Nanoparticle-immersed paper imprinting mass spectrometry imaging reveals uptake and translocation mechanism of pesticides in plants

Design and discovery of carrier-mediated modified pesticides are vital for reducing pesticide dosage and increasing utilization,yet it remains a great challenge due to limited insights into plant translocation mechanisms.Nanostructure/nanoparticle assisted laser desorption/ionization strategy has established itself as a preferential analytical tool for biological tissue analysis,whereas potential applications in plant sciences are hindered with regard to the inability to slice plant leaves and petals.Herein,we report gold nanoparticle(AuNP)-immersed paper imprinting mass spectrometry imaging(MSI)for the spatiotemporal visualization of pesticide translocation in plant leaves.This approach plays a dual role in preserving spatial information and improving ionization efficiency for pesticides regardless of imaging artifacts due to homogenous AuNP deposition.Using this MSI platform,we proposed the elaborate plant translocation mechanism of agrochemicals for the first time,which is currently poorly understood.The dynamic processes of carrier-mediated pesticides can be clearly visualized,including crossing of plasma membranes by transporters,translocation downward in stems through the phloem,diffusion to the xylem and,conversely,accumulation at margins of the treated leaves.Moreover,this AuNP-assisted paper imprinting method could be highly compatible with laser-based MSI instruments,expediting researches across a broad range of fields,especially in nanomaterial development and life sciences.

A temporo-spatial pharmacometabolomics method to characterize pharmacokinetics and pharmacodynamics in the brain microregions by using ambient mass spectrometry imaging

The brain is the most advanced organ with various complex structural and functional microregions. It is often challenging to understand what and where the molecular events would occur for a given drug treatment in the brain. Herein, a temporo-spatial pharmacometabolomics method was proposed based on ambient mass spectrometry imaging and was applied to evaluate the microregional effect of olanzapine(OLZ) on brain tissue and demonstrate its effectiveness in characterizing the microregional pharmacokinetics and pharmacodynamics of OLZ for improved understanding of the molecular mechanism of drugs acting on the microregions of the brain. It accurately and simultaneously illustrated the levels dynamics and microregional distribution of various substances, including exogenous drugs and its metabolites, as well as endogenous functional metabolites from complicated brain tissue. The targeted imaging analysis of the prototype drug and its metabolites presented the absorption, distribution, metabolism, and excretion characteristics of the drug itself. Moreover, the endogenous functional metabolites were identified along with the associated therapeutic and adverse effects of the drug, which can reflect the pharmacodynamics effect on the microregional brain. Therefore, this method is significant in elucidating and understanding the molecular mechanism of central nervous system drugs at the temporo and spatial metabolic level of system biology.

On-tissue chemical derivatization enables spatiotemporal heterogeneity visualization of oxylipins in esophageal cancer xenograft via ambient mass spectrometry imaging

On-tissue chemical derivatization(OTCD)effectively enhances ionization efficiency of low abundant and poorly ionized functional molecules to improve detection sensitivity and coverage of mass spectrometry imaging(MSI).Combination OTCD and MSI provides a novel strategy for visualizing previously undisclosed metabolic heterogeneity in tumor.Herein,we present a method to visualize heterogeneous metabolism of oxylipins within tumor by coupling OTCD with airflow-assisted desorption electrospray ionization(AFADESI)-MSI.Taking Girard’s P as a derivatization reagent,easily ionized hydrazide and quaternary amine groups were introduced into the structure of carbonyl metabolites via condensation reaction.Oxylipins,including 127 fatty aldehydes(FALs)and 71 oxo fatty acids(FAs),were detected and imaged in esophageal cancer xenograft with AFADESI-MSI after OTCD.Then t-distributed stochastic neighbor embedding and random forest were exploited to precisely locate the distribution of oxylipins in heterogeneous tumor tissue.With this method,we surprisingly found almost all FALs and oxo FAs significantly accumulated in the core region of tumor,and exhibited a gradual increase trend in tumor over time.These results reveal spatiotemporal heterogeneity of oxylipins in tumor progression,highlighting the value of OTCD combined with MSI to gain deeper insights into understanding tumor metabolism.

Mass Spectrometry and Mass Spectrometry Imaging-based Thyroid Cancer Analysis

Thyroid carcinoma is one of the most common endocrine malignant diseases worldwide.With the rapid development of medical technology,early and effective diagnostic methods could be able to improve the survival rate and quality of life of patients suffering from the disease.Considering the complexity of cancer,some specific detection method is desired for diagnosis and treatment.Mass spectrometry imaging(MSI)is an emerging technique for acquiring molecular information from biological tissues without staining and labeling,including qualitative,quantitative and spatial distribution information.Over the past several decades,MSI has been widely used for pharmacological monitoring,biomolecular imaging of cells and tissues.In this review,we introduce the tumor progression and histological characteristics of thyroid cancer,and focus mainly on the preparation of biological specimens for MSI and mass spectrometry(MS)analysis,as well as the recent progress in MS and MSI-based thyroid cancer research.This review thoroughly discusses the importance of MS and MSI for clinical diagnosis,identification and prognosis of thyroid cancer,and provides some new clues for molecular mechanisms research and tumor metastasis.