Biosynthetic investigation of ?-lactones in Sextonia rubra wood using in situ TOF-SIMS MS/MS imaging to localize and characterize biosynthetic intermediates.
ABSTRACT: Molecular analysis by parallel tandem mass spectrometry (MS/MS) imaging contributes to the in situ characterization of biosynthetic intermediates which is crucial for deciphering the metabolic pathways in living organisms. We report the first use of TOF-SIMS MS/MS imaging for the cellular localization and characterization of biosynthetic intermediates of bioactive ?-lactones rubrynolide and rubrenolide in the Amazonian tree Sextonia rubra (Lauraceae). Five ?-lactones, including previously reported rubrynolide and rubrenolide, were isolated using a conventional approach and their structural characterization and localization at a lateral resolution of ~400?nm was later achieved using TOF-SIMS MS/MS imaging analysis. 2D/3D MS imaging at subcellular level reveals that putative biosynthetic ?-lactones intermediates are localized in the same cell types (ray parenchyma cells and oil cells) as rubrynolide and rubrenolide. Consequently, a revised metabolic pathway of rubrynolide was proposed, which involves the reaction between 2-hydroxysuccinic acid and 3-oxotetradecanoic acid, contrary to previous studies suggesting a single polyketide precursor. Our results provide insights into plant metabolite production in wood tissues and, overall, demonstrate that combining high spatial resolution TOF-SIMS imaging and MS/MS structural characterization offers new opportunities for studying molecular and cellular biochemistry in plants.
Project description:Lipids are abundant biomolecules performing central roles to maintain proper functioning of cells and biological bodies. Due to their highly complex composition, it is critical to obtain information of lipid structures in order to identify particular lipids which are relevant for a biological process or metabolic pathway under study. Among currently available molecular identification techniques, MS/MS in secondary ion mass spectrometry (SIMS) imaging has been of high interest in the bioanalytical community as it allows visualization of intact molecules in biological samples as well as elucidation of their chemical structures. However, there have been few applications using SIMS and MS/MS owing to instrumental challenges for this capability. We performed MS and MS/MS imaging to study the lipid structures of Drosophila brain using the J105 and 40-keV Ar<sub>4000</sub><sup>+</sup> gas cluster ion source, with the novelty being the use of MS/MS SIMS analysis of intact lipids in the fly brain. Glycerophospholipids were identified by MS/MS profiling. MS/MS was also used to characterize diglyceride fragment ions and to identify them as triacylglyceride fragments. Moreover, MS/MS imaging offers a unique possibility for detailed elucidation of biomolecular distribution with high accuracy based on the ion images of its fragments. This is particularly useful in the presence of interferences which disturb the interpretation of biomolecular localization. Graphical abstract MS/MS was performed during time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis of Drosophila melongaster (fruit fly) to elucidate the structure and origin of different chemical species in the brain including a range of different phospholipid classes (PC, PI, PE) and di- and triacylglycerides (DAG & TAG) species where reference MS/MS spectra provided a potential means of discriminating between the isobaric [M-OH]<sup>+</sup> ion of DAGs and the [M-RCO]<sup>+</sup> ion of TAGs.
Project description: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.
Project description:A unique method for identification of biomolecular components in different biological specimens, while preserving the capability for high speed 2D and 3D molecular imaging, is employed to investigate cellular response to oxidative stress. The employed method enables observing the distribution of the antioxidant α-tocopherol and other molecules in cellular structures via time-of-flight secondary ion mass spectrometry (TOF-SIMS (MS<sup>1</sup>)) imaging in parallel with tandem mass spectrometry (MS<sup>2</sup>) imaging, collected simultaneously. The described method is employed to examine a network formed by neuronal cells differentiated from human induced pluripotent stem cells (iPSCs), a model for investigating human neurons in vitro. The antioxidant α-tocopherol is identified in situ within different cellular layers utilizing a 3D TOF-SIMS tandem MS imaging analysis. As oxidative stress also plays an important role in mediating inflammation, the study was expanded to whole body tissue sections of M. marinum-infected zebrafish, a model organism for tuberculosis. The TOF-SIMS tandem MS imaging results reveal an increased presence of α-tocopherol in response to the pathogen. Graphical Abstract ᅟ.
Project description:Coenzyme A (CoA) is an essential cofactor for dozens of reactions in intermediary metabolism. Dysregulation of CoA synthesis or acyl CoA metabolism can result in metabolic or neurodegenerative disease. Although several methods use liquid chromatography coupled with mass spectrometry/mass spectrometry (LC-MS/MS) to quantify acyl CoA levels in biological samples, few allow for simultaneous measurement of intermediates in the CoA biosynthetic pathway. Here we describe a simple sample preparation and LC-MS/MS method that can measure both short-chain acyl CoAs and biosynthetic precursors of CoA. The method does not require use of a solid phase extraction column during sample preparation and exhibits high sensitivity, precision, and accuracy. It reproduces expected changes from known effectors of cellular CoA homeostasis and helps clarify the mechanism by which excess concentrations of etomoxir reduce intracellular CoA levels.
Project description: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.
Project description:Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a surface analysis technique well-suited to detect and identify trace surface species. With the latest analyzers, ion sources and data analysis methods, imaging ToF-SIMS provides detailed 2-D and 3-D surface reactivity maps. Coupling multivariate analysis methods such as principal component analysis (PCA) with ToF-SIMS provides a powerful method for differentiating spatial regions with different chemistries. ToF-SIMS and PCA are used in this study to image and analyze a two-component photolithograph-patterned surface chemistry currently published and commercialized for bioassays, bio-chips and cell-based biosensors. A widely used reactive surface coupling chemistry, N-hydroxysuccinimide (NHS), and 2-methoxyethylamine (MeO) were co-patterned into adjacent regions on a commercial microarray polymer coating using standard photolithography methods involving deposition, patterning and removal of a routinely used photoresist material. After routine processing, ToF-SIMS and PCA of the patterned surface revealed significant residual photoresist material remaining at the interface of the NHS/MeO patterns, as well as lower concentrations of residual photoresist material remaining within the MeO-containing regions, providing spatial mapping and residue analysis not evident from other characterization techniques. As detection of surface photoresist residue remains an inherent challenge in photolithographic processing of a wide array of materials, the use of ToF-SIMS coupled with PCA is shown to be a high-resolution characterization tool with the high sensitivity and specificity required for surface quality control measurements following photolithography and pattern development relevant to many current processes.
Project description:The biomolecular imaging of cell-nanoparticle (NP) interactions using time-of-flight secondary ion mass spectrometry (ToF-SIMS) represents an evolving tool in nanotoxicology. In this study we present the three dimensional (3D) distribution of nanomaterials within biomolecular agglomerates using ToF-SIMS imaging. This novel approach was used to model the resistance of human alveolar A549 cells against gold (Au) ion toxicity through intra- and extracellular biomineralization. At low Au concentrations (?1?mM HAuCl4) 3D-ToF-SIMS imaging reveals a homogenous intracellular distribution of Au-NPs in combination with polydisperse spherical NPs biomineralized in different layers on the cell surface. However, at higher precursor concentrations (?2?mM) supplemented with biogenic spherical NPs as seeds, cells start to biosynthesize partially embedded long aspect ratio fiber-like Au nanostructures. Most interestingly, A549 cells seem to be able to sense the enhanced Au concentration. They change the chemical composition of the extracellular NP agglomerates from threonine-O-3-phosphate aureate to an arginine-Au(I)-imine. Furthermore they adopt the extracellular mineralization process from spheres to irregular structures to nanoribbons in a dose-dependent manner with increasing Au concentrations. The results achieved regarding size, shape and chemical specificity were cross checked by SEM-EDX and single particle (sp-)ICP-MS. Our findings demonstrate the potential of ToF-SIMS 3D imaging to better understand cell-NP interactions and their impact in nanotoxicology.
Project description:Time of flight secondary ion mass spectrometry (ToF-SIMS) is a powerful surface-sensitive characterization tool allowing the imaging of chemical properties over a wide range of organic and inorganic material systems. This technique allows precise studies of chemical composition with sub-100-nm lateral and nanometer depth spatial resolution. However, comprehensive interpretation of ToF-SIMS results is challenging because of the very large data volume and high dimensionality. Furthermore, investigation of samples with pronounced topographical features is complicated by systematic and measureable shifts in the mass spectrum. In this work we developed an approach for the interpretation of the ToF-SIMS data, based on the advanced data analytics. Along with characterization of the chemical composition, our approach allows extraction of the sample surface morphology from a time of flight registration technique. This approach allows one to perform correlated investigations of surface morphology, biological function, and chemical composition of Arabidopsis roots.
Project description:Secondary ion mass spectrometry is a powerful method for imaging biological samples with high spatial resolution. Whole section time-of-flight-secondary ion mass spectrometry (TOF-SIMS) scans and multivariate data analysis have been performed on the human spinal cord in order to delineate anatomical regions of interest based on their chemical distribution pattern. TOF-SIMS analysis of thoracic spinal cord sections was performed at 5 ?m resolution within 2 h. Multivariate image analysis by means of principal component analysis and maximum auto correlation factor analysis resulted in detection of more than 400 m/z peaks that were found to be significantly changed. Here, the results show characteristic biochemical distributions that are well in line with major histological regions, including gray and white matter. As an approach for iterative segmentation, we further evaluated previously outlined regions of interest as identified by multivariate image analysis. Here, further discrimination of the gray matter into ventral, lateral, and dorsal neuroanatomical regions was observed. TOF-SIMS imaging has been carried out at submicrometer resolution obtaining localization and characterization of spinal motor neurons based on their chemical fingerprint, including neurotransmitter precursors that serve as molecular indicators for motor neuron integrity. Thus, TOF-SIMS can be used as an approach for chemical histology and pathology. TOF-SIMS holds immense potential for investigating the subcellular mechanisms underlying spinal cord related diseases including chronic pain and amyotrophic lateral sclerosis.
Project description:Asian lacquer is a special polymeric material tapped from lacquer trees. The tree's sap is a complex mixture of compounds, such as catechol lipids, polysaccharides, glycoproteins, enzymes, and water. Researchers have not yet quantitatively analyzed blended lacquers. We evaluated the compositions of Japanese and Vietnamese lacquers, and blends of the two, using time-of-flight secondary ion mass spectrometry (ToF-SIMS), pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), and high-performance liquid chromatography (HPLC). ToF-SIMS provided quantitative results for blended lacquers; provided structural information on polymeric lacquer films; and indicated the presence of dimers of urushiol-urushiol, urushiol-laccol, and laccol-laccol derivatives. We used Py-GC/MS and HPLC to obtain linear calibration curves. The specific peak intensity was a linear function of the ratio of Japanese to Vietnamese lacquer in the blends. For an unknown mixture, all three techniques gave essentially the same results. These quantitative methods will be useful for improving the physical properties of polymeric lacquer films, and evaluating the lacquer quality in industry and historic conservation.