RESUMO
Molecular analysis on the single-cell level represents a rapidly growing field in the life sciences. While bulk analysis from a pool of cells provides a general molecular profile, it is blind to heterogeneities between individual cells. This heterogeneity, however, is an inherent property of every cell population. Its analysis is fundamental to understanding the development, function, and role of specific cells of the same genotype that display different phenotypical properties. Single-cell mass spectrometry (MS) aims to provide broad molecular information for a significantly large number of cells to help decipher cellular heterogeneity using statistical analysis. Here, we present a sensitive approach to single-cell MS based on high-resolution MALDI-2-MS imaging in combination with MALDI-compatible staining and use of optical microscopy. Our approach allowed analyzing large amounts of unperturbed cells directly from the growth chamber. Confident coregistration of both modalities enabled a reliable compilation of single-cell mass spectra and a straightforward inclusion of optical as well as mass spectrometric features in the interpretation of data. The resulting multimodal datasets permit the use of various statistical methods like machine learning-driven classification and multivariate analysis based on molecular profile and establish a direct connection of MS data with microscopy information of individual cells. Displaying data in the form of histograms for individual signal intensities helps to investigate heterogeneous expression of specific lipids within the cell culture and to identify subpopulations intuitively. Ultimately, t-MALDI-2-MSI measurements at 2-µm pixel sizes deliver a glimpse of intracellular lipid distributions and reveal molecular profiles for subcellular domains.
Assuntos
Imagem Molecular , Análise de Célula Única , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Técnicas de Cultura de Células , Metabolismo dos Lipídeos , Imagem Molecular/métodos , Análise Multivariada , Análise de Célula Única/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
The successful application of matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) in pharmaceutical research is strongly dependent on the detection of the drug of interest at physiologically relevant concentrations. Here we explored how insufficient sensitivity due to low ionization efficiency and/or the interaction of the drug molecule with the local biochemical environment of the tissue can be mitigated for many compound classes using the recently introduced MALDI-MSI coupled with laser-induced postionization, known as MALDI-2-MSI. Leveraging a MALDI-MSI screen of about 1,200 medicines/drug-like compounds from a broad range of medicinal application areas, we demonstrate a significant improvement in drug detection and the degree of sensitivity uplift by using MALDI-2 versus traditional MALDI. Our evaluation was made under simulated imaging conditions using liver homogenate sections as substrate, onto which the compounds were spotted to mimic biological conditions to the first order. To enable an evaluable detection by both MALDI and MALDI-2 for the majority of employed compounds, we spotted 1 µL of a 10 mM solution using a spotting robot and performed our experiments with a Bruker timsTOF fleX MALDI-2 instrument in both positive and negative ion modes. Specifically, we demonstrate using a large cohort of drug-like compounds that â¼60% of the tested compounds showed a more than 10-fold increase in signal intensity and â¼16% showed a more than 100-fold increase upon use of MALDI-2 postionization. Such increases in sensitivity could help advance pharmaceutical MALDI-MSI applications toward the single-cell level.
Assuntos
Fígado , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Preparações Farmacêuticas/análise , Preparações Farmacêuticas/química , Fígado/química , Avaliação Pré-Clínica de MedicamentosRESUMO
The precise fatty acyl chain configuration of cardiolipin (CL), a tetrameric mitochondrial-specific membrane lipid, exhibits dependence on cell and tissue types. A powerful method to map CL profiles in tissue sections in a spatially resolved manner is matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). To build on and explore this potential, we employed a quadrupole time-of-flight mass spectrometer along with optimized sample preparation protocols. We imaged the CL profiles of individual murine retinal cell layers at a pixel size of 10 µm. In combination with tandem MS, we obtained detailed insights into the CL composition of individual retinal cell layers. In particular, we found differential expression of the polyunsaturated fatty acids (PUFA) linoleic, arachidonic, and docosahexaenoic acids. PUFAs are prone to peroxidation and hence regarded as critical factors in development and progression of retinal pathologies, such as age-related macular degeneration (AMD). The ability of MALDI-MSI to provide cues on the CL composition in neuronal tissue with close to single-cell resolution can provide important insights into retinal physiology in health and disease.
Assuntos
Cardiolipinas , Retina , Animais , Camundongos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cardiolipinas/análise , Retina/química , Diagnóstico por Imagem , Manejo de EspécimesRESUMO
This Feature focuses on a review of recent developments in mass spectrometry imaging (MSI) of lipid isomers in biological tissues. The tandem MS techniques utilizing online and offline chemical derivatization procedures, ion activation techniques such as ozone-induced dissociation (OzID), ultraviolet photodissociation (UVPD), or electron-induced dissociation (EID), and other techniques such as coupling of ion mobility with MSI are discussed. The importance of resolving lipid isomers in diseases is highlighted.
Assuntos
Ozônio , Isomerismo , Lipídeos/análise , Espectrometria de Massas/métodos , Ozônio/química , Raios UltravioletaRESUMO
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a rapidly growing method in the life sciences. However, for many analyte classes, its sensitivity is limited due to poor ionization efficiencies. To mitigate this problem, we here introduce a novel post-ionization scheme based on single-photon induced chemical ionization using pulsed RF-Kr lamps. The fine-vacuum conditions of a dual ion-funnel ion source effectively thermalize the evolving MALDI plume and enable ample gas-phase reactions. Injected chemical dopants crucially support fragment-less ionization to [M+H]+ /[M-H]- species. Based on this interplay, numerous glycerophospho-, sphingo-, and further lipids, registered from mammalian tissue sections, were boosted by up to three orders of magnitude, similar to results obtained with laser-based post-ionization (MALDI-2). Experiments with deuterated matrix and dopant, however, indicated complex chemical ionization pathways different from MALDI-2.
Assuntos
Diagnóstico por Imagem , Lasers , Animais , Mamíferos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) makes it possible to simultaneously visualize the spatial distribution of dozens to hundreds of different biomolecules (e.g., phospho- and glycolipids) in tissue sections and in cell cultures. The implementation of novel desorption and (post-)ionization techniques has recently pushed the pixel size of this imaging technique to the low micrometer scale and below and thus to a cellular and potentially sub-cellular level. However, to fully exploit this potential for cell biology and biomedicine, sample preparation becomes highly demanding. Here, we investigated the effect of several key parameters on the quality of the sample preparation and achievable spatial resolution, that include the washing, drying, chemical fixation, and matrix coating steps. The incubation of cells with formalin for about 5 min in combination with isotonic washing and mild drying produced a robust protocol that largely preserved not only cell morphologies, but also the molecular integrities of amine group-containing cell membrane phospholipids (phosphatidylethanolamines and -serines). A disadvantage of the chemical fixation is an increased permeabilization of cell membranes, resulting in leakage of cytosolic compounds. We demonstrate the pros and cons of the protocols with four model cell lines, cultured directly on indium tin oxide (ITO)-coated glass slides. Transmission (t-)mode MALDI-2-MSI enabled on a Q Exactive plus Orbitrap mass spectrometer was used to analyze the cultures at a pixel size of 2 µm. Phase contrast light microscopy and scanning electron microscopy were used as complementary methods. The protocols described could prove to be an important contribution to the advancement of single-cell MALDI imaging, especially for the characterization of cell-to-cell heterogeneities at a molecular level.
Assuntos
Formaldeído , Fosfolipídeos , Linhagem Celular , Diagnóstico por Imagem , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Waminoa sp. acoel flatworms hosting Symbiodiniaceae and the related Amphidinium dinoflagellate algae are an interesting model system for symbiosis in marine environments. While the host provides a microhabitat and safety, the algae power the system by photosynthesis and supply the worm with nutrients. Among these nutrients are sterols, including cholesterol and numerous phytosterols. While it is widely accepted that these compounds are produced by the symbiotic dinoflagellates, their transfer to and fate within the sterol-auxotrophic Waminoa worm host as well as their role in its metabolism are unknown. Here we used matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging combined with laser-induced post-ionization and trapped ion mobility spectrometry (MALDI-2-TIMS-MSI) to map the spatial distribution of over 30 different sterol species in sections of the symbiotic system. The use of laser post-ionization crucially increased ion yields and allowed the recording of images with a pixel size of 5 µm. Trapped ion mobility spectrometry (TIMS) helped with the tentative assignment of over 30 sterol species. Correlation with anatomical features of the worm, revealed by host-derived phospholipid signals, and the location of the dinoflagellates, revealed by chlorophyll a signal, disclosed peculiar differences in the distribution of different sterol species (e.g. of cholesterol versus stigmasterol) within the receiving host. These findings point to sterol species-specific roles in the metabolism of Waminoa beyond a mere source of energy. They also underline the value of the MALDI-2-TIMS-MSI method to future research in the spatially resolved analysis of sterols.
Assuntos
Dinoflagellida/química , Platelmintos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Esteróis/análise , Animais , Dinoflagellida/fisiologia , Espectrometria de Mobilidade Iônica/métodos , Platelmintos/fisiologia , Esteróis/metabolismo , SimbioseRESUMO
N-glycans are important players in a variety of pathologies including different types of cancer, (auto)immune diseases, and also viral infections. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an important tool for high-throughput N-glycan profiling and, upon use of tandem MS, for structure determination. By use of MALDI-MS imaging (MSI) in combination with PNGase F treatment, also spatially correlated N-glycan profiling from tissue sections becomes possible. Here we coupled laser-induced postionization, or MALDI-2, to a trapped ion mobility quadrupole time-of-flight mass spectrometer (timsTOF fleX MALDI-2, Bruker Daltonics). We demonstrate that with MALDI-2 the sensitivity for the detection of molecular [M - H]- species of N-glycans increased by about 3 orders of magnitude. Compared to the current gold standard, the positive ion mode analysis of [M + Na]+ adducts, a sensitivity increase by about a factor of 10 is achieved. By exploiting the advantageous fragmentation behavior of [M - H]- ions, exceedingly rich structural information on the composition of complex N-glycans was moreover obtained directly from thin tissue sections of human cerebellum and upon use of low-energy collision-induced dissociation tandem MS. In another set of experiments, in this case by use of a modified Synapt G2-S QTOF mass spectrometer (Waters), we investigated the influence of relevant input parameters, in particular pressure of the N2 cooling gas in the ion source, delay between the two laser pulses, and that of their pulse energies. In this way, analytical conditions were identified at which molecular ion abundances were maximized and fragmentation reactions minimized. The use of negative ion mode MALDI-2-MSI could constitute a valuable tool in glycobiology research.
Assuntos
Polissacarídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Idoso de 80 Anos ou mais , Encéfalo/metabolismo , Encéfalo/patologia , Humanos , Íons/química , Masculino , Polissacarídeos/química , Razão Sinal-RuídoRESUMO
Matrix-assisted laser desorption/ionization combined with laser-induced postionization (MALDI-2) is a recently introduced method for enhanced mass spectrometry imaging of numerous classes of biomolecules, including phospho- and glycolipids in tissue sections at high lateral resolution. Here we describe the first adaptation of the technology to a Bruker timsTOF fleX mass spectrometer. Upon use of a 1 kHz postionization laser, MALDI-2 produces a sizable increase in the number of detected features as well as in ion signal intensities. This enhancement is similar to that described previously for low repetition rate MALDI-2 systems, but now enables substantially enhanced measurement speeds. In our proof-of-concept study, we furthermore demonstrate, on examples of rat brain and testis tissue sections, that the combination of MALDI-2 with the trapped ion mobility spectrometry (TIMS) functionality of the instrument can crucially support unravelling the complex molecular composition of the lipidome. Numerous isomeric/isobaric ion species are successfully separated upon using the collisional cross section (CCS) as additional specific physical property. With the possibilities of high data acquisition speed or high separation powers in combination with the increased sensitivity of MALDI-2 available in one instrument, the described methodology could be a valuable tool in many areas of biological and medical research.
Assuntos
Lipídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Encéfalo/metabolismo , Espectrometria de Mobilidade Iônica , Lipídeos/química , Masculino , Ratos , Testículo/metabolismoRESUMO
The main cellular receptors of Shiga toxins (Stxs), the neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer/CD77) and globotetraosylceramide (Gb4Cer), are significantly upregulated in about half of the human colorectal carcinomas (CRC) and in other cancers. Therefore, conjugates exploiting the Gb3Cer/Gb4Cer-binding B subunit of Stx (StxB) have attracted great interest for both diagnostic and adjuvant therapeutic interventions. Moreover, fucosylated GSLs were recognized as potential tumor-associated targets. One obstacle to a broader use of these receptor/ligand systems is that the contribution of specific GSLs to tumorigenesis, in particular, in the context of an altered lipid metabolism, is only poorly understood. A second is that also nondiseased organs (e.g., kidney) and blood vessels can express high levels of certain GSLs, not least Gb3Cer/Gb4Cer. Here, we used, in a proof-of-concept study, matrix-assisted laser desorption/ionization mass spectrometry imaging combined with laser-induced postionization (MALDI-2-MSI) to simultaneously visualize the distribution of several Gb3Cer/Gb4Cer lipoforms and those of related GSLs (e.g., Gb3Cer/Gb4Cer precursors and fucosylated GSLs) in tissue biopsies from three CRC patients. Using MALDI-2 and StxB-based immunofluorescence microscopy, Gb3Cer and Gb4Cer were mainly found in dedifferentiated tumor cell areas, tumor stroma, and tumor-infiltrating blood vessels. Notably, fucosylated GSL such as Fuc-(n)Lc4Cer generally showed a highly localized expression in dysplastic glands and indian file-like cells infiltrating adipose tissue. Our "molecular histology" approach could support stratifying patients for intratumoral GSL expression to identify an optimal therapeutic strategy. The improved chemical coverage by MALDI-2 can also help to improve our understanding of the molecular basis of tumor development and GSL metabolism.
Assuntos
Neoplasias do Colo/diagnóstico , Glicoesfingolipídeos/análise , Estudos de Coortes , Humanos , Imuno-Histoquímica , Microscopia de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Visualizing the differential distribution of carbon-carbon double bond (CâC db) positional isomers of unsaturated phospholipids (PL) in tissue sections by use of refined matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) technologies offers a high promise to deeper understand PL metabolism and isomer-specific functions in health and disease. Here we introduce an on-tissue ozonization protocol that enables a particular straightforward derivatization of unsaturated lipids in tissue sections. Collision-induced dissociation (CID) of MALDI-generated ozonide ions (with yields in the several ten percent range) produced the Criegee fragment ion pairs, which are indicative of CâC db position(s). We used our technique for visualizing the differential distribution of Δ9 and Δ11 isomers of phosphatidylcholines in mouse brain and in human colon samples with the desorption laser spot size 15 µm, emphasizing the potential of the technique to expose local isomer-specific metabolism of PLs.
Assuntos
Ozônio/química , Fosfolipídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Carbono/química , Colo/diagnóstico por imagem , Colo/metabolismo , Humanos , Íons/química , Isomerismo , Camundongos , Fosfolipídeos/metabolismoRESUMO
Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is increasingly used to visualize the chemical communication between microorganisms. However, to fully exploit the potential of this label-free technique, crucial methodological advances are still needed. In particular, with current microbial MALDI-MSI methods chemical coverage is strongly limited to well ionizing compounds and a safe MSI-compatible inactivation of microbial viability and quenching of metabolism is not possible. Here, we introduce a membrane-based culturing workflow that enables a rapid MSI-compatible steam inactivation of pathogens and generation of a flat surface. We equipped precision mass spectrometers with laser-postionization (MALDI-2) modules to increase the analytical sensitivity by up to several orders of magnitude. In this way, for example 39 different 2-alkylquinolones with differential expression patterns and a similar number of glycerophospholipids were simultaneously visualized from single cultures of Pseudomonas aeruginosa at about 50 µm resolution. To visualize the metabolic exchange between competing microorganisms, we challenged commensal Escherichia coli MG1655 and virulence factor-depleted E. coli C600 strains with enteropathogenic Shiga-toxin negative E. coli O26:H11, and Staphylococcus aureus with antagonistic P. aeruginosa. Insight into the three-dimensional organization of a biofilm of the probiotic E. coli Nissle 1917 at 15 µm pixel size was obtained after developing an embedding/cryosectioning protocol. Our advanced protocols could help to substantially increase the application range of microbial MS imaging.
Assuntos
Microbiota/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Biofilmes , Comunicação Celular , Escherichia coli , Imagem Molecular/métodos , Imagem Óptica/métodos , Pseudomonas aeruginosa , Staphylococcus aureusRESUMO
Long-chain cuticular hydrocarbons (CHC) are key components of chemical communication in many insects. The parasitoid jewel wasps from the genus Nasonia use their CHC profile as sex pheromone and for species recognition. The standard analytical tool to analyze CHC is gas chromatography coupled with mass spectrometric detection (GC/MS). This method reliably identifies short- to long-chain alkanes and alkenes, but CHC with more than 40 carbon atoms are usually not detected. Here, we applied two laser mass spectrometry (MS) techniques, namely direct laser desorption/ionization (d)LDI and silver-assisted (Ag-)LDI MS, respectively, to analyze CHC profiles of N. vitripennis, N. giraulti, and N. longicornis directly from the cuticle or extracts. Furthermore, we applied direct analysis in real-time (DART) MS as another orthogonal technique for extracts. The three methods corroborated previous results based on GC/MS, i.e., the production of CHC with carbon numbers between C25 and C40. However, we discovered a novel series of long-chain CHC ranging from C41 to C51/C52. Additionally, several previously unreported singly and doubly unsaturated alkenes in the C31-C39 range were found. Use of principal component analysis (PCA) revealed that the composition of the newly discovered CHC varies significantly between species, sex, and age of the animals. Our study adds to the growing literature on the presence of very long-chain CHC in insects and hints at putative roles in insect communication. Graphical abstract.
Assuntos
Hidrocarbonetos/análise , Espectrometria de Massas/métodos , Atrativos Sexuais/análise , Vespas/química , Alcenos/análise , Escamas de Animais/química , Animais , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Masculino , Análise de Componente PrincipalRESUMO
Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) visualizes the distribution of phospho- and glycolipids in tissue sections. However, C=C double-bond (db) positional isomers generally cannot be distinguished. Now an on-tissue Paternò-Büchi (PB) derivatization procedure that exploits benzaldehyde as a MALDI-MSI-compatible reagent is introduced. Laser-induced postionization (MALDI-2) was used to boost the yields of protonated PB products. Collision-induced dissociation of these species generated characteristic ion pairs, indicative of C=C position, for numerous singly and polyunsaturated phospholipids and glycosphingolipids in mouse brain tissue. Several db-positional isomers of phosphatidylcholine and phosphatidylserine species were expressed with highly differential levels in the white and gray matter areas of cerebellum. Our PB-MALDI-MS/MS procedure could help to better understand the physiological role of these db-positional isomers.
Assuntos
Carbono/química , Glicolipídeos/química , Fosfolipídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Benzaldeídos/química , Encéfalo/metabolismo , Camundongos , SuínosRESUMO
The interplay between the wavelength of the laser and the absorption profile of the matrix constitutes a crucial factor in matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Numerous studies have shown that typically best analytical results are obtained if the laser wavelength matches the UV absorption band of the matrix in the solid state well. However, many powerful matrices exhibit peak absorptions which differ notably from the standard MALDI laser wavelengths of 337, 349, and 355 nm, respectively. Here we used two wavelength-tunable lasers to investigate the MALDI wavelength dependence with a selected set of such matrices. We studied 3-hydroxypicolinic acid (3-HPA), 2,4,6-trihydroxyacetophenon (THAP), dithranol (1,8-dihydroxy-10H-anthracen-9-on), 2-(4'-hydroxybenzeneazo)benzoic acid (HABA), and 6-aza-2-thiothymine (ATT). For analyte systems we investigated DNA oligomers (3-HPA), phospholipids (dithranol, THAP, HABA), and non-covalent peptide-peptide and protein-peptide complexes (ATT). We recorded analyte ion and total ion counts as a function of wavelength and laser fluence between 213 and 600 nm. Although the so-generated comprehensive heat maps generally corroborated the previously made findings, several fine features became notable. For example, despite a still high optical absorption exhibited by some of the matrices in the visible wavelength range, ion yields generally dropped strongly, indicating a change in ionization mechanism. Moreover, the non-covalent complexes were optimally detected at wavelengths corresponding to a relatively low optical absorptivity of the ATT matrix, presumably because of ejection of a particular cold MALDI plume. Our comprehensive data shed useful light into the MALDI mechanisms and could assist in further methodological advancement of the technique.
RESUMO
Silver-assisted laser desorption/ionization mass spectrometry can be used for the analysis of small molecules. For example, adduct formation with silver cations enables the molecular analysis of long-chain hydrocarbons, which are difficult to ionize via conventional matrix-assisted laser desorption ionization (MALDI). Here we used highly porous silver foils, produced by etching with nitric acid, as sample substrates for LDI mass spectrometry. As model system for the analysis of complex lipid mixtures, cuticular extracts of fruit flies (Drosophila melanogaster) and worker bees (Apis mellifera) were investigated. The mass spectra obtained by spotting extract onto the etched silver substrates demonstrate the sensitive detection of numerous lipid classes such as long-chain saturated and unsaturated hydrocarbons, fatty acyl alcohols, wax esters, and triacylglycerols. MS imaging of cuticular surfaces with a lateral resolution of a few tens of micrometers became possible after blotting, i.e., after transferring lipids by physical contact with the substrate. The examples of pheromone-producing male hindwings of the squinting bush brown butterfly (Bicyclus anynana) and a fingermark are shown. Because the substrates are also easy to produce, they provide a viable alternative to colloidal silver nanoparticles and other so far described silver substrates.
Assuntos
Lasers , Lipídeos/isolamento & purificação , Prata/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Abelhas/química , Drosophila melanogaster/química , Lipídeos/química , Nanopartículas Metálicas/químicaRESUMO
The fungus Claviceps purpurea produces highly toxic ergot alkaloids and accumulates these in the hardened bodies of fungal mycelium. These so-called sclerotia, or ergot bodies, replace the crop seed of infected plants, which can include numerous important food- and feedstuff such as rye and wheat. While several studies have explored details of the infection process and development of ergot bodies, little information is available on the spatial distribution of the mycotoxins in the sclerotia. Here we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) at a lateral resolution of 35 µm to visualize the distribution of two representative alkaloids, ergocristine and ergometrine, produced by Ecc93 and Gal 310 variants of C. purpurea, respectively, after infection of rye. To improve cryosectioning of this fragile biological material tissue with complex texture, we developed a practical embedding protocol based on cellulose polymers. The MALDI-MS images recorded from the so produced intact tissues sections revealed that ergometrine exhibited a relatively homogeneous distribution throughout the ergot body, whereas ergocristine was found to be enriched in the proximal region. This finding can be correlated to the morphological development of sclerotia as ergot alkaloids are only produced in the sphacelial stage. The ability to localize toxins and other secondary metabolites in intact sections of crop-infecting fungi with high lateral resolution renders MALDI-MSI a powerful tool for investigating biosynthetic pathways and for obtaining a deeper understanding of the parasite-host interaction. Graphical abstract Workflow for identification and spatial localization of ergot alkaloids in infected rye grains.
Assuntos
Claviceps/química , Alcaloides de Claviceps/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to simultaneously visualize the lateral distribution of different lipid classes in tissue sections, but the applicability of the method to real-life samples is often limited by ion suppression effects. In particular, the presence of abundant phosphatidylcholines (PCs) can reduce the ion yields for all other lipid species in positive ion mode measurements. Here, we used on-tissue treatment with buffer-free phospholipase C (PLC) to near-quantitatively degrade PCs in fresh-frozen tissue sections. The ion signal intensities of mono-, di-, and oligohexosylceramides were enhanced by up to 10-fold. In addition, visualization of Shiga toxin receptor globotriaosylceramide (Gb3Cer) in the kidneys of wild-type and α-galactosidase A-knockout (Fabry) mice was possible at about ten micrometer resolution. Importantly, the PLC treatment did not decrease the high lateral resolution of the MS imaging analysis.
Assuntos
Encéfalo/enzimologia , Rim/enzimologia , Glicoesfingolipídeos Neutros/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fosfolipases Tipo C/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Glicoesfingolipídeos Neutros/metabolismo , Fosfolipases Tipo C/químicaRESUMO
Arsenic-containing lipids (arsenolipids) are natural products of marine organisms such as fish, invertebrates, and algae, many of which are important seafoods. A major group of arsenolipids, namely, the arsenic-containing hydrocarbons (AsHC), have recently been shown to be cytotoxic to human liver and bladder cells, a result that has stimulated interest in the chemistry and toxicology of these compounds. In this study, elemental laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS) and molecular matrix-assisted laser desorption/ionization (MALDI-)MS were used to image and quantify the uptake of an AsHC in the model organism Drosophila melanogaster. Using these two complementary methods, both an enrichment of arsenic and the presence of the AsHC in the brain were revealed, indicating that the intact arsenolipid had crossed the blood-brain barrier. Simultaneous acquisition of quantitative elemental concentrations and molecular distributions could allow new insight into organ-specific enrichment and possible transportation processes of arsenic-containing bioactive compounds in living organisms.