RESUMO
In an era when population aging is increasing the burden of neurodegenerative conditions, deciphering the mechanisms underlying brain senescence is more important than ever. Here, we present a spatial metabolomics analysis of age-induced neurochemical alterations in the mouse brain using negative ionization mode mass spectrometry imaging. The age-dependent effects of the acetylcholinesterase inhibitor tacrine were simultaneously examined. For ultrahigh mass resolution analysis, we utilized a Fourier-transform ion cyclotron resonance spectrometer. To complement this, a trapped ion mobility spectrometry time-of-flight analyzer provided high speed and lateral resolution. The chosen approach facilitated the detection and identification of a wide range of metabolites, from amino acids to sphingolipids. We reported significant, age-dependent alterations in brain lipids which were most evident for sulfatides and lysophosphatidic acids. Sulfatide species, which are mainly localized to white matter, either increased or decreased with age, depending on the carbon chain length and hydroxylation stage. Lysophosphatidic acids were found to decrease with age in the detailed cortical and hippocampal subregions. An age-dependent increase in the glutamine/glutamate ratio, an indicator of glia-neuron interconnection and neurotoxicity, was detected after tacrine administration. The presented metabolic mapping approach was able to provide visualizations of the lipid signaling and neurotransmission alterations induced by early aging and can thus be beneficial to further elucidating age-related neurochemical pathways.
Assuntos
Envelhecimento , Metabolômica , Animais , Metabolômica/métodos , Envelhecimento/metabolismo , Envelhecimento/efeitos dos fármacos , Camundongos , Tacrina/farmacologia , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Inibidores da Colinesterase/farmacologia , Lisofosfolipídeos/metabolismo , Sulfoglicoesfingolipídeos/metabolismo , Espectrometria de Massas/métodosRESUMO
Perfluorooctanoic acid (PFOA) is a synthetic perfluorinated chemical classified as a persistent organic pollutant. PFOA has been linked to many toxic effects, including liver injury. Many studies report that PFOA exposure alters serum and hepatic lipid metabolism. However, lipidomic pathways altered by PFOA exposure are largely unknown and only a few lipid classes, mostly triacylglycerol (TG), are usually considered in lipid analysis. Here, we performed a global lipidomic analysis on the liver of PFOA-exposed (high-dose and short-duration) and control mice by combining three mass spectrometry (MS) techniques: liquid chromatography with tandem mass spectrometry (LC-MS/MS), matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Among all hepatic lipids identified by LC-MS/MS analysis, more than 350 were statistically impacted (increased or decreased levels) after PFOA exposure, as confirmed by multi-variate data analysis. The levels of many lipid species from different lipid classes, most notably phosphatidylethanolamine (PE), phosphatidylcholine (PC), and TG, were significantly altered. Subsequent lipidomic analysis highlights the pathways significantly impacted by PFOA exposure, with the glycerophospholipid metabolism being the most impacted, and the changes in the lipidome network, which connects all the lipid species together. MALDI-MSI displays the heterogeneous distribution of the affected lipids and PFOA, revealing different areas of lipid expression linked to PFOA localization. TOF-SIMS localizes PFOA at the cellular level, supporting MALDI-MSI results. This multi-modal MS analysis unveils the lipidomic impact of PFOA in the mouse liver after high-dose and short-term exposure and opens new opportunities in toxicology.
Assuntos
Lipidômica , Espectrometria de Massas em Tandem , Camundongos , Animais , Cromatografia Líquida , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Caprilatos , Triglicerídeos , FígadoRESUMO
Mass spectrometry imaging is a technique uniquely suited to localize and identify lipids in a tissue sample. Using an atmospheric pressure (AP-) matrix-assisted laser desorption ionization (MALDI) source coupled to an Orbitrap Elite, numerous lipid locations and structures can be determined in high mass resolution spectra and at cellular spatial resolution, but careful sample preparation is necessary. We tested 11 protocols on serial brain sections for the commonly used MALDI matrices CHCA, norharmane, DHB, DHAP, THAP, and DAN in combination with tissue washing and matrix additives to determine the lipid coverage, signal intensity, and spatial resolution achievable with AP-MALDI. In positive-ion mode, the most lipids could be detected with CHCA and THAP, while THAP and DAN without additional treatment offered the best signal intensities. In negative-ion mode, DAN showed the best lipid coverage and DHAP performed superiorly for gangliosides. DHB produced intense cholesterol signals in the white matter. One hundred fifty-five lipids were assigned in positive-ion mode (THAP) and 137 in negative-ion mode (DAN), and 76 peaks were identified using on-tissue tandem-MS. The spatial resolution achievable with DAN was 10 µm, confirmed with on tissue line-scans. This enabled the association of lipid species to single neurons in AP-MALDI images. The results show that the performance of AP-MALDI is comparable to vacuum MALDI techniques for lipid imaging.
Assuntos
Pressão Atmosférica , Lipídeos , Encéfalo , Lipídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Basal cell carcinoma (BCC) is the most common skin malignancy. In fact, it is as common as the sum of all other skin malignancies combined and the incidence is rising. In this focused and histology-guided study, tissue from a patient diagnosed with aggressive BCC was analyzed by imaging mass spectrometry in order to probe the chemistry of the complex tumor environment. Time-of-flight secondary ion mass spectrometry using a (CO2)6 k + gas cluster ion beam allowed a wide range of lipid species to be detected. Their distributions were then imaged in the tissue that contained small tumor islands that were histologically classified as more/less aggressive. Maximum autocorrelation factor (MAF) analysis highlighted chemical differences between the tumors and the surrounding stroma. A closer inspection of the distribution of individual ions, selected based on the MAF loadings, showed heterogeneity in signal between different microtumors, suggesting the potential of chemically grading the aggressiveness of each individual tumor island. Sphingomyelin lipids were found to be located in stroma containing inflammatory cells.
Assuntos
Carcinoma Basocelular/patologia , Lipídeos/análise , Neoplasias Cutâneas/patologia , Espectrometria de Massa de Íon Secundário , Biomarcadores Tumorais/análise , Carcinoma Basocelular/metabolismo , Humanos , Análise de Componente Principal , Neoplasias Cutâneas/metabolismo , Microambiente TumoralRESUMO
Lipids have been recognized as key players in cell signaling and disease. Information on their location and distribution within a biological system, under varying conditions, is necessary to understand the contributions of different lipid species to an altered phenotype. Imaging mass spectrometry techniques, such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) and matrix-assisted laser desorption/ionization (MALDI), are capable of revealing global lipid distributions in tissues in an untargeted fashion. However, to confidently identify the species present in a sample, orthogonal analyses like tandem MS (MS/MS) are often required. This can be accomplished by bulk sample analysis with liquid chromatography (LC)-MS/MS, which can provide confident lipid identifications, at the expense of losing location-specific information. Here, using planarian flatworms as a model system, we demonstrate that imaging gas cluster ion beam (GCIB)-ToF-SIMS has the unique capability to simultaneously detect, identify, and image lipid species with subcellular resolution in tissue sections. The parallel detection of both, intact lipids and their respective fragments, allows for unique identification of some species without the need of performing an additional orthogonal MS/MS analysis. This was accomplished by correlating intact lipid and associated fragment SIMS images. The lipid assignments, respective fragment identities, and locations gathered from ToF-SIMS data were confirmed via LC-MS/MS on lipid extracts and ultrahigh mass resolution MALDI-MS imaging. Together, these data show that the semidestructive nature of ToF-SIMS can be utilized advantageously to enable both confident molecular annotations and to determine the locations of species within a biological sample.
Assuntos
Lipídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massa de Íon Secundário/métodos , Animais , Cromatografia Líquida , Humanos , Metabolismo dos Lipídeos , Espectrometria de Massas em Tandem/métodos , Distribuição TecidualRESUMO
Planarian flatworms are known as the masters of regeneration, re-growing an entire organism from as little as 1/279th part of their body. While the proteomics of these processes has been studied extensively, the planarian lipodome remains relatively unknown. In this study we investigate the lipid profile of planarian tissue sections with imaging Time-of-Flight - Secondary-Ion-Mass-Spectrometry (ToF-SIMS). ToF-SIMS is a label-free technique capable of gathering intact, location specific lipid information on a cellular scale. Lipid identities are confirmed using LC-MS/MS. Our data shows that different organ structures within planarians have unique lipid profiles. The 22-carbon atom poly unsaturated fatty acids (PUFAs) which occur in unusually high amounts in planarians are found to be mainly located in the testes. Additionally, we observe that planarians contain various odd numbered fatty acid species, that are usually found in bacteria, localized in the reproductive and ectodermal structures of the planarian. An abundance of poorly understood ether fatty acids and ether lipids were found in unique areas in planarians as well as a new, yet unidentified class of potential lipids in planarian intestines. Identifying the location of these lipids in the planarian body provides insights into their bodily functions and, in combination with knowledge about their diet and their genome, enables drawing conclusions about planarian fatty acid processing.
Assuntos
Lipídeos/análise , Planárias/química , Planárias/ultraestrutura , Animais , Ácidos Graxos/análise , Microscopia , Imagem Óptica , Planárias/anatomia & histologia , Espectrometria de Massa de Íon SecundárioRESUMO
Breast cancer is an umbrella term used to describe a collection of different diseases with broad inter- and intratumor heterogeneity. Understanding this variation is critical in order to develop, and precisely prescribe, new treatments. Changes in the lipid metabolism of cancerous cells can provide important indications as to the metabolic state of the cells but are difficult to investigate with conventional histological methods. Due to the introduction of new higher energy (40 kV) gas cluster ion beams (GCIBs), time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging is now capable of providing information on the distribution of hundreds of molecular species simultaneously on a cellular to subcellular scale. GCIB-ToF-SIMS was used to elucidate changes in lipid composition in nine breast cancer biopsy samples. Improved molecular signal generation by the GCIB produced location-specific information that revealed elevated levels of essential lipids to be related to inflammatory cells in the stroma, while cancerous areas were dominated by nonessential fatty acids and a variety of phosphatidylinositol species with further in-tumor variety arising from decreased desaturase activity. These changes in lipid composition due to different enzyme activity are seemingly independent of oxygen availability and can be linked to favorable cell membrane properties for either proliferation/invasion or drug resistance/survival.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Ácidos Graxos/análise , Lipídeos/análise , Imagem Óptica , Microambiente Tumoral , Argônio/química , Feminino , Fulerenos/química , Humanos , Íons/química , Espectrometria de Massa de Íon SecundárioRESUMO
Neurons communicate via an essential process called exocytosis. Cholesterol, an abundant lipid in both secretory vesicles and cell plasma membrane can affect this process. In this study, amperometric recordings of vesicular dopamine release from two different artificial cell models created from a giant unilamellar liposome and a bleb cell plasma membrane, show that with higher membrane cholesterol the kinetics for vesicular release are decelerated in a concentration dependent manner. This reduction in exocytotic speed was consistent for two observed modes of exocytosis, full and partial release. Partial release events, which only occurred in the bleb cell model due to the higher tension in the system, exhibited amperometric spikes with three distinct shapes. In addition to the classic transient, some spikes displayed a current ramp or plateau following the maximum peak current. These post spike features represent neurotransmitter release from a dilated pore before constriction and show that enhancing membrane rigidity via cholesterol adds resistance to a dilated pore to re-close. This implies that the cholesterol dependent biophysical properties of the membrane directly affect the exocytosis kinetics and that membrane tension along with membrane rigidity can influence the fusion pore dynamics and stabilization which is central to regulation of neurochemical release.
Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Exocitose/fisiologia , Neurônios/metabolismo , Animais , Neurônios/citologia , Células PC12 , RatosRESUMO
Escherichia coli is able to rapidly adjust the biophysical properties of its membrane phospholipids to adapt to environmental challenges including starvation stress. These membrane lipid modifications were investigated in glucose starved E. coli cultures and compared to a ΔrelAΔspoT (ppGpp(0)) mutant strain of E. coli, deficient in the stringent response, by means of time-of-flight-secondary ion mass spectrometry (TOF-SIMS). Recent advances in TOF-SIMS, through the implementation of gas cluster ion beams (GCIBs), now permit the analysis of higher mass species from native, underivatized, biological specimen, i.e., intact bacterial cells. Cultures in stationary phase were found to exhibit a radically different lipid composition as compared to cultures in the exponential growth phase. Wild-type E. coli reacted upon carbon starvation by lipid modifications including elongation, cyclopropanation, and increased cardiolipin formation. Observations are consistent with variants of cardiolipins (CL), phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidic acids (PA), and fatty acids. Notably, despite having a proteomic profile and a gene expression profile somewhat similar to the wild-type during growth, the ppGpp(0) mutant E. coli strain was found to exhibit modified phospholipids corresponding to unsaturated analogues of those found in the wild-type. We concluded that the ppGpp(0) mutant reacts upon starvation stress by elongation and desaturation of fatty acyl chains, implying that only the last step of the lipid modification, the cyclopropanation, is under stringent control. These observations suggest alternative stress response mechanisms and illustrate the role of the RelA and SpoT enzymes in the biosynthetic pathway underlying these lipid modifications.
Assuntos
Cardiolipinas/química , Escherichia coli/isolamento & purificação , Ácidos Graxos/química , Ácidos Fosfatídicos/química , Fosfatidiletanolaminas/química , Fosfatidilgliceróis/química , Espectrometria de Massa de Íon Secundário , Fatores de TempoRESUMO
Lipidomics has been an expanding field since researchers began to recognize the signaling functions of lipids and their involvement in disease. Time-of-flight secondary ion mass spectrometry is a valuable tool for studying the distribution of a wide range of lipids in multiple brain regions, but in order to make valuable scientific contributions, one has to be aware of the influence that sample treatment can have on the results. In this article, the authors discuss different sample treatment protocols for rodent brain sections focusing on signal from the hippocampus and surrounding areas. The authors compare frozen hydrated analysis to freeze drying, which is the standard in most research facilities, and reactive vapor exposure (trifluoroacetic acid and NH3). The results show that in order to preserve brain chemistry close to a native state, frozen hydrated analysis is the most suitable, but execution can be difficult. Freeze drying is prone to produce artifacts as cholesterol migrates to surface, masking other signals. This effect can be partially reversed by exposing freeze dried sections to reactive vapor. When analyzing brain sections in negative ion mode, exposing those sections to NH3 vapor can re-establish the diversity in lipid signal found in frozen hydrated analyzed sections. This is accomplished by removing cholesterol and uncovering sulfatide signals, allowing more anatomical regions to be visualized.
Assuntos
Hipocampo/anatomia & histologia , Hipocampo/química , Lipídeos/análise , Manejo de Espécimes/métodos , Espectrometria de Massa de Íon Secundário/métodos , Animais , Ratos ZuckerRESUMO
In secondary ion mass spectrometry (SIMS), the beneficial effect of cesium implantation or flooding on the enhancement of negative secondary ion yields has been investigated in detail for various semiconductor and metal samples. All results have been obtained for monatomic ion bombardment. Recent progress in SIMS is based to a large extent on the development and use of cluster primary ions. In this work we show that the enhancement of negative secondary ions induced by the combination of ion bombardment with simultaneous cesium flooding is valid not only for monatomic ion bombardment but also for cluster primary ions. Experiments carried out using C60+ and Ar4000+ bombardment on silicon show that yields of negative secondary silicon ions can be optimized in the same way as by Ga+ and Cs+ bombardment. Both for monatomic and cluster ion bombardment, the optimization does not depend on the primary ion species. Hence, it can be assumed that the silicon results are also valid for other cluster primary ions and that results obtained for monatomic ion bombardment on other semiconductor and metal samples are also valid for cluster ion bombardment. In SIMS, cluster primary ions are also largely used for the analysis of organic matter. For polycarbonate, our results show that Ar4000+ bombardment combined with cesium flooding enhances secondary ion signals by a factor of 6. This can be attributed to the removal of charging effects and/or reduced fragmentation, but no major influence on ionization processes can be observed. The use of cesium flooding for the imaging of cells was also investigated and a significant enhancement of secondary ion yields was observed. Hence, cesium flooding has also a vast potential for SIMS analyses with cluster ion bombardment.
RESUMO
We report the results of a VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study on the measurement of composition in organic depth profiling. Layered samples with known binary compositions of Irganox 1010 and either Irganox 1098 or Fmoc-pentafluoro-l-phenylalanine in each layer were manufactured in a single batch and distributed to more than 20 participating laboratories. The samples were analyzed using argon cluster ion sputtering and either X-ray photoelectron spectroscopy (XPS) or time-of-flight secondary ion mass spectrometry (ToF-SIMS) to generate depth profiles. Participants were asked to estimate the volume fractions in two of the layers and were provided with the compositions of all other layers. Participants using XPS provided volume fractions within 0.03 of the nominal values. Participants using ToF-SIMS either made no attempt, or used various methods that gave results ranging in error from 0.02 to over 0.10 in volume fraction, the latter representing a 50% relative error for a nominal volume fraction of 0.2. Error was predominantly caused by inadequacy in the ability to compensate for primary ion intensity variations and the matrix effect in SIMS. Matrix effects in these materials appear to be more pronounced as the number of atoms in both the primary analytical ion and the secondary ion increase. Using the participants' data we show that organic SIMS matrix effects can be measured and are remarkably consistent between instruments. We provide recommendations for identifying and compensating for matrix effects. Finally, we demonstrate, using a simple normalization method, that virtually all ToF-SIMS participants could have obtained estimates of volume fraction that were at least as accurate and consistent as XPS.
Assuntos
Laboratórios , Compostos Orgânicos/química , Espectroscopia Fotoeletrônica , Espectrometria de Massa de Íon Secundário , Hidroxitolueno Butilado/análogos & derivados , Hidroxitolueno Butilado/química , Fluorenos/química , Fluorbenzenos/químicaRESUMO
Imaging mass spectrometry has shown to be a valuable method in medical research and can be performed using different instrumentation and sample preparation methods, each one with specific advantages and drawbacks. Time-of-flight-secondary ion mass spectrometry (TOF-SIMS) has the advantage of high spatial resolution imaging but is often restricted to low mass molecular signals and can be very sensitive to sample preparation artifacts. In this report we demonstrate the advantages of using gas cluster ion beams (GCIBs) in combination with trifluoracetic acid (TFA) vapor exposure for the imaging of lipids in mouse brain sections. There is an optimum exposure to TFA that is beneficial for increasing high mass signal as well as producing signal from previously unobserved species in the mass spectrum. Cholesterol enrichment and crystallization on the sample surface is removed by TFA exposure uncovering a wider range of lipid species in the white matter regions of the tissue, greatly expanding the chemical coverage and the potential application of TOF-SIMS imaging in neurological studies. Ar4000(+) (40 keV) in combination with TFA treatment facilitates high resolution, high mass imaging closing the gap between TOF-SIMS and matrix-assisted laser desorption ionization (MALDI).
Assuntos
Gases/química , Lipídeos/análise , Imagem Molecular , Ácido Trifluoracético/química , Animais , Encéfalo , Íons/química , Ratos , Espectrometria de Massa de Íon Secundário , Fatores de Tempo , VolatilizaçãoRESUMO
Several pathways modulating longevity and stress resistance converge on translation by targeting ribosomal proteins or initiation factors, but whether this involves modifications of ribosomal RNA is unclear. Here, we show that reduced levels of the conserved RNA methyltransferase NSUN5 increase the lifespan and stress resistance in yeast, worms and flies. Rcm1, the yeast homologue of NSUN5, methylates C2278 within a conserved region of 25S rRNA. Loss of Rcm1 alters the structural conformation of the ribosome in close proximity to C2278, as well as translational fidelity, and favours recruitment of a distinct subset of oxidative stress-responsive mRNAs into polysomes. Thus, rather than merely being a static molecular machine executing translation, the ribosome exhibits functional diversity by modification of just a single rRNA nucleotide, resulting in an alteration of organismal physiological behaviour, and linking rRNA-mediated translational regulation to modulation of lifespan, and differential stress response.
Assuntos
Metilação , RNA Ribossômico/genética , Animais , Drosophila , Feminino , Organismos Hermafroditas/genética , Organismos Hermafroditas/fisiologia , Humanos , Expectativa de Vida , Masculino , Camundongos , RNA Ribossômico/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologiaRESUMO
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is an important technique for studying chemical composition of micrometer scale objects due to its high spatial resolution imaging capabilities and chemical specificity. In this work we focus on the application of ToF-SIMS to gain insight into the chemistry of micrometer size liposomes as a potential model for neurotransmitter vesicles. Two models of giant liposomes were analyzed: histamine and aqueous two phase system (ATPS)-containing liposomes. Characterization of the internal structure of single fixed liposomes was done both with the Bi3+ and C60+ ion sources. The depth profiling capability of ToF-SIMS was used to investigate the liposome interior.