MALDI MS Imaging of Chickpea Seeds (Cicer arietinum) and Crab's Eye Vine (Abrus precatorius) after Tryptic Digestion Allows Spatially Resolved Identification of Plant Proteins.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) imaging following in situ enzymatic digestion is a versatile analytical method for the untargeted investigation of protein distributions, which has rarely been used for plants so far. The present study describes a workflow for in situ tryptic digestion of plant seed tissue for MALDI MS imaging. Substantial modifications to the sample preparation procedure for mammalian tissues were necessary to cater to the specific properties of plant materials. For the first time, distributions of tryptic peptides were successfully visualized in plant tissue using MS imaging with accurate mass detection. Sixteen proteins were visualized and identified in chickpea seeds showing different distribution patterns, e.g., in the cotyledons, radicle, or testa. All tryptic peptides were detected with a mass resolution higher than 60,000 as well as a mass accuracy better than 1.5 ppm root-mean-square error and were matched to results from complementary liquid chromatography-MS/MS (LC-MS/MS) data. The developed method was also applied to crab's eye vine seeds for targeted MS imaging of the toxic protein abrin, showing the presence of abrin-a in all compartments. Abrin (59 kDa), as well as the majority of proteins visualized in chickpeas, was larger than 50 kDa and would thus not be readily accessible by top-down MS imaging. Since antibodies for plant proteins are often not readily available, in situ digestion MS imaging provides unique information, as it makes the distribution and identification of larger proteins in plant tissues accessible in an untargeted manner. This opens up new possibilities in the field of plant science as well as to assess the nutritional quality and/or safety of crops.

Interleukin-13-Overexpressing Mice Represent an Advanced Preclinical Model for Detecting the Distribution of Antimycobacterial Drugs within Centrally Necrotizing Granulomas.

The Mycobacterium tuberculosis-harboring granuloma with a necrotic center surrounded by a fibrous capsule is the hallmark of tuberculosis (TB). For a successful treatment, antibiotics need to penetrate these complex structures to reach their bacterial targets. Hence, animal models reflecting the pulmonary pathology of TB patients are of particular importance to improve the preclinical validation of novel drug candidates. M. tuberculosis-infected interleukin-13-overexpressing (IL-13tg) mice develop a TB pathology very similar to patients and, in contrast to other mouse models, also share pathogenetic mechanisms. Accordingly, IL-13tg animals represent an ideal model for analyzing the penetration of novel anti-TB drugs into various compartments of necrotic granulomas by matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MS imaging). In the present study, we evaluated the suitability of BALB/c IL-13tg mice for determining the antibiotic distribution within necrotizing lesions. To this end, we established a workflow based on the inactivation of M. tuberculosis by gamma irradiation while preserving lung tissue integrity and drug distribution, which is essential for correlating drug penetration with lesion pathology. MALDI-MS imaging analysis of clofazimine, pyrazinamide, and rifampicin revealed a drug-specific distribution within different lesion types, including cellular granulomas, developing in BALB/c wild-type mice, and necrotic granulomas in BALB/c IL-13tg animals, emphasizing the necessity of preclinical models reflecting human pathology. Most importantly, our study demonstrates that BALB/c IL-13tg mice recapitulate the penetration of antibiotics into human lesions. Therefore, our workflow in combination with the IL-13tg mouse model provides an improved and accelerated evaluation of novel anti-TB drugs and new regimens in the preclinical stage.

Do Anti-tuberculosis Drugs Reach Their Target?─High-Resolution Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Provides Information on Drug Penetration into Necrotic Granulomas.

Tuberculosis (TB) is characterized by mycobacteria-harboring centrally necrotizing granulomas. The efficacy of anti-TB drugs depends on their ability to reach the bacteria in the center of these lesions. Therefore, we developed a mass spectrometry (MS) imaging workflow to evaluate drug penetration in tissue. We employed a specific mouse model that─in contrast to regular inbred mice─strongly resembles human TB pathology. Mycobacterium tuberculosis was inactivated in lung sections of these mice by γ-irradiation using a protocol that was optimized to be compatible with high spatial resolution MS imaging. Different distributions in necrotic granulomas could be observed for the anti-TB drugs clofazimine, pyrazinamide, and rifampicin at a pixel size of 30 µm. Clofazimine, imaged here for the first time in necrotic granulomas of mice, showed higher intensities in the surrounding tissue than in necrotic granulomas, confirming data observed in TB patients. Using high spatial resolution drug and lipid imaging (5 µm pixel size) in combination with a newly developed data analysis tool, we found that clofazimine does penetrate to some extent into necrotic granulomas and accumulates in the macrophages inside the granulomas. These results demonstrate that our imaging platform improves the predictive power of preclinical animal models. Our workflow is currently being applied in preclinical studies for novel anti-TB drugs within the German Center for Infection Research (DZIF). It can also be extended to other applications in drug development and beyond. In particular, our data analysis approach can be used to investigate diffusion processes by MS imaging in general.

Classification of target tissues of Eisenia fetida using sequential multimodal chemical analysis and machine learning.

Acquiring comprehensive knowledge about the uptake of pollutants, impact on tissue integrity and the effects at the molecular level in organisms is of increasing interest due to the environmental exposure to numerous contaminants. The analysis of tissues can be performed by histological examination, which is still time-consuming and restricted to target-specific staining methods. The histological approaches can be complemented with chemical imaging analysis. Chemical imaging of tissue sections is typically performed using a single imaging approach. However, for toxicological testing of environmental pollutants, a multimodal approach combined with improved data acquisition and evaluation is desirable, since it may allow for more rapid tissue characterization and give further information on ecotoxicological effects at the tissue level. Therefore, using the soil model organism Eisenia fetida as a model, we developed a sequential workflow combining Fourier transform infrared spectroscopy (FTIR) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) for chemical analysis of the same tissue sections. Data analysis of the FTIR spectra via random decision forest (RDF) classification enabled the rapid identification of target tissues (e.g., digestive tissue), which are relevant from an ecotoxicological point of view. MALDI imaging analysis provided specific lipid species which are sensitive to metabolic changes and environmental stressors. Taken together, our approach provides a fast and reproducible workflow for label-free histochemical tissue analyses in E. fetida, which can be applied to other model organisms as well.

Matrix ions as internal standard for high mass accuracy matrix-assisted laser desorption/ionization mass spectrometry imaging.

RATIONALE: High mass accuracy is indispensable for reliable identification in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) imaging. Ubiquitous matrix ions can serve as reference masses for mass calibration if their sum formula is known. Here we report an overview of ions generated on tissue by 11 common MALDI matrices for use in internal or external mass calibration. METHODS: Matrices covered in this study were applied onto coronal mouse brain sections using a pneumatic sprayer setup. MALDI imaging was performed on a Q Exactive HF orbital trapping mass spectrometer coupled to an AP-SMALDI 10 source. Measurements were conducted with high mass resolution (240 k full width at half maximum at m/z 200) and high mass accuracy with a root mean square mass error of better than 1.5 ppm achieved via internal mass calibration using matrix ions. RESULTS: MALDI MS imaging was used to investigate ions generated on tissue by 11 common MALDI matrices. An example of using matrix ions for internal mass calibration in MALDI imaging of drug substances and lipids in murine lung sections is presented. Tables containing the cluster composition, sum formulae, and the measured and theoretical m/z ratios of the identified ions were compiled for each matrix. CONCLUSION: Using matrix ions as reference masses for internal and external mass calibration in MALDI MS imaging is an effective and elegant way to achieve sub-ppm mass accuracy as it makes use of ubiquitous signals present in every MALDI MS spectrum without the need for an additional calibration standard.

Error-Free Data Visualization and Processing through imzML and mzML Validation.

Open data formats are key to facilitating data processing, sharing, and integration. The imzML format ( http://imzml.org/ ) has drastically improved these aspects of mass spectrometry imaging data. Efficient processing of data depends on data sets which are consistent and adhere to the specifications; however, this is not always the case. Here we present a validation tool for data stored in both imzML and the HUPO-PSI mass spectrometery counterpart, mzML, to identify any deviations from the published (i)mzML standard which could cause issues for the user when visualizing or processing data. The tool is released in two forms, a graphical user interface (GUI) for ease of use, and a command line version to fit into existing workflows and pipelines. When certain known issues are encountered, such as the presence of negative values for the location of the binary data, the validator resolves the issue automatically upon saving. The GUI version of the validator also allows editing of the metadata included within the (i)mzML files in order to resolve inconsistencies. We also present a means of performing conditional validation on the metadata within (i)mzML files, where user-defined rules are validated against depending on whether specific metadata are present (or not). For example, if the MALDI term is present, then additional rules related to MALDI (such as the requirement of inclusion of laser parameters) can be validated against this. This enables a flexible and more thorough automated validation of (i)mzML data. Such a system is necessary for validating data against more comprehensive sets of metadata such as minimum reporting guidelines or metadata requirements prior to submission and acceptance of data to data repositories. We demonstrate how this tool can be used to validate against the proposed minimum reporting guidelines in MSI as well as institute specific metadata criteria. The validator tool is endorsed for validation of imzML ( http://imzml.org/ ) and mzML ( http://www.psidev.info/mzml ) and is made available through the respective Web sites. The validator is also released as open source under Mozilla Public License 2.0 at https://gitlab.com/imzML/imzMLValidator .

Approaching cellular resolution and reliable identification in mass spectrometry imaging of tryptic peptides.

On-tissue digestion has become the preferred method to identify proteins in mass spectrometry (MS) imaging. In this study, we report advances in data acquisition and protein identification for MS imaging after on-tissue digestion. Tryptic peptides in a coronal mouse brain section were measured at 50 µm pixel size and revealed detailed histological structures, e.g., the ependyma (consisting of one to two cell layers), which was confirmed by H&E staining. This demonstrates that MS imaging of tryptic peptides at or close to cellular resolution is within reach. We also describe a detailed identification workflow which resulted in the identification of 99 proteins (with 435 corresponding peptides), based on comparison with LC-MS/MS data and in silico digest. These results were obtained with stringent parameters, including high mass accuracy in imaging mode (RSME < 3 ppm) and at least two unique peptides per protein showing consistent spatial distribution. We identified almost 50% of proteins with at least four corresponding peptides. As there is no agreed approach for identification of proteins after on-tissue digestion yet, we discuss our workflow in detail and make the corresponding mass spectral data available as "open data" via ProteomeXchange (identifier PXD003172). With this, we would like to contribute to a more effective discussion and the development of new approaches for tryptic peptide identification in MS imaging. From an experimental point of view, we demonstrate the improvement due to the combination of high spatial resolution and high mass resolution/mass accuracy on a measurement at 25 µm pixel size in mouse cerebellum tissue. A whole body section of a mouse pub imaged at 50 µm pixel size (40 GB, 230,000 spectra) demonstrates the stability of our protocol. For this data set, we developed a workflow that is based on conversion to the common data format imzML and sequential application of freely available software tools. In combination, the presented results for spatial resolution, protein identification, and data processing constitute significant improvements for the field of on-tissue digestion. Graphical abstract MS imaging of coronal mouse brain cerebellum with a pixel size of 25 µm: A Optical image, B myelin staining, C H&E staining, and D MS image overlay (RGB) of tryptic peptides m/z = 726.4045 ± 0.005, HGFLPR + H+ (red), m/z = 536.3173 ± 0.005, AKPAK + Na+ (green), and m/z = 994.5436 ± 0.005, WRQLIEK + Na+ (blue).

Mass Spectrometry Imaging of the Hypoxia Marker Pimonidazole in a Breast Tumor Model.

Although tumor hypoxia is associated with tumor aggressiveness and resistance to cancer treatment, many details of hypoxia-induced changes in tumors remain to be elucidated. Mass spectrometry imaging (MSI) is a technique that is well suited to study the biomolecular composition of specific tissue regions, such as hypoxic tumor regions. Here, we investigate the use of pimonidazole as an exogenous hypoxia marker for matrix-assisted laser desorption/ionization (MALDI) MSI. In hypoxic cells, pimonidazole is reduced and forms reactive products that bind to thiol groups in proteins, peptides, and amino acids. We show that a reductively activated pimonidazole metabolite can be imaged by MALDI-MSI in a breast tumor xenograft model. Immunohistochemical detection of pimonidazole adducts on adjacent tissue sections confirmed that this metabolite is localized to hypoxic tissue regions. We used this metabolite to image hypoxic tissue regions and their associated lipid and small molecule distributions with MALDI-MSI. We identified a heterogeneous distribution of 1-methylnicotinamide and acetylcarnitine, which mostly colocalized with hypoxic tumor regions. As pimonidazole is a widely used immunohistochemical marker of tissue hypoxia, it is likely that the presented direct MALDI-MSI approach is also applicable to other tissues from pimonidazole-injected animals or humans.

Natural products in Glycyrrhiza glabra (licorice) rhizome imaged at the cellular level by atmospheric pressure matrix-assisted laser desorption/ionization tandem mass spectrometry imaging.

The rhizome of Glycyrrhiza glabra (licorice) was analyzed by high-resolution mass spectrometry imaging and tandem mass spectrometry imaging. An atmospheric pressure matrix-assisted laser desorption/ionization imaging ion source was combined with an orbital trapping mass spectrometer in order to obtain high-resolution imaging in mass and space. Sections of the rhizome were imaged with a spatial resolution of 10 µm in the positive ion mode, and a large number of secondary metabolites were localized and identified based on their accurate mass and MS/MS fragmentation patterns. Major tissue-specific metabolites, including free flavonoids, flavonoid glycosides and saponins, were successfully detected and visualized in images, showing their distributions at the cellular level. The analytical power of the technique was tested in the imaging of two isobaric licorice saponins with a mass difference of only 0.02 Da. With a mass resolving power of 140 000 and a bin width of 5 ppm in the image processing, the two compounds were well resolved in full-scan mode, and appeared with different distributions in the tissue sections. The identities of the compounds and their distributions were validated in a subsequent MS/MS imaging experiment, thereby confirming their identities and excluding possible analyte interference. The use of high spatial resolution, high mass resolution and tandem mass spectrometry in imaging experiments provides significant information about the biosynthetic pathway of flavonoids and saponins in legume species, combing the spatially resolved chemical information with morphological details at the microscopic level. Furthermore, the technique offers a scheme capable of high-throughput profiling of metabolites in plant tissues.

Phospholipid Topography of Whole-Body Sections of the Anopheles stephensi Mosquito, Characterized by High-Resolution Atmospheric-Pressure Scanning Microprobe Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

High-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) has been employed to study the molecular anatomical structure of rodent malaria vector Anopheles stephensi mosquitoes. A dedicated sample preparation method was developed which suits both, the special tissue properties of the sample and the requirements of high-resolution MALDI imaging. Embedding in 5% carboxymethylcellulose (CMC) was used to maintain the tissue integrity of the whole mosquitoes, being very soft, fragile, and difficult to handle. Individual lipid compounds, specifically representing certain cell types, tissue areas, or organs, were detected and imaged in 20 µm-thick whole-body tissue sections at a spatial resolution of 12 µm per image pixel. Mass spectrometric data and information quality were based on a mass resolution of 70,000 (at m/z 200) and a mass accuracy of better than 2 ppm in positive-ion mode on an orbital trapping mass spectrometer. A total of 67 imaged lipids were assigned by database search and, in a number of cases, identified via additional MS/MS fragmentation studies directly from tissue. This is the first MSI study at 12 µm spatial resolution of the malaria vector Anopheles. The study provides insights into the molecular anatomy of Anopheles stephensi and the distribution and localization of major classes of glycerophospholipids and sphingolipids. These data can be a basis for future experiments, investigating, e.g., the metabolism of Plasmodium-infected and -uninfected Anopheles mosquitoes.

Resolution pattern for mass spectrometry imaging.

RATIONALE: Up to now, there is no 'gold standard' for determining the resolution of a mass spectrometry imaging (MSI) setup (comprising the instrument, the sample preparation, the sample and the instrument settings). A standard sample in combination with a standard protocol to define the MSI resolution would be desirable in order to compare the setups of different laboratories, and as a regular quality control/performance check. METHODS: Microstructured resolution patterns were fabricated that can be used to determine the spatial resolution in MSI experiments, down to the range of a few µm. Two different strategies were employed, one where the resolution pattern is laser machined into a thin metal foil, which can be placed over a sample to be imaged, and a second one where hydrophilic grooves are machined into an omniphobic coating covering the surface of an indium tin oxide covered glass slide. When dragging a sample solution over the slide's surface, the sample is automatically retained in the hydrophilic grooves, but repelled by the omniphobic coating. RESULTS: The technology was tested on a commercial matrix-assisted laser desorption/ionization (MALDI) imaging instrument, and a spatial resolution in the vicinity of 50 µm was determined. The finest features of the microstructured resolution patterns are compatible with the best spatial resolution of MALDI imaging systems available to date. CONCLUSIONS: The use of metal resolution grids or glass slides with hydrophilic/hydrophobic structures is suitable for the convenient determination of the resolution limit of the MALDI imaging instrument as determined by its hardware. These structures are straightforward both to produce and to use.

Uptake and bioavailability of anthocyanins and phenolic acids from grape/blueberry juice and smoothie in vitro and in vivo.

The goal of eating five servings of fruits and vegetables a day has not yet been achieved. The intake of polyphenols such as anthocyanins (ACN) could be improved by consuming smoothies and juices that are increasingly popular, especially in children; however, bioavailability data concerning food matrix effects are scarce. Thus, we conducted a randomised, cross-over, bioavailability study (n 10) to determine the bioavailability of ACN and their metabolites from an ACN-rich grape/blueberry juice (841 mg ACN/litre) and smoothie (983 mg ACN/litre) in vivo, and the uptake of a corresponding grape/blueberry extract in vitro. After the intake of beverage (0·33 litres), plasma and fractionated urine samples were collected and analysed by ultra-performance liquid chromatography coupled to MS. The most abundant ACN found in plasma and urine were malvidin and peonidin as native ACN and as glucuronidated metabolites as well as 3,4-dihydroxybenzoic acid (3,4-DHB); minor ACN (delphinidin, cyanidin and petunidin) were only detected as native glycosides. Plasma pharmacokinetics and recoveries of urinary metabolites of ACN were not different for juice or smoothie intake; however, the phenolic acid 3,4-DHB was significantly better bioavailable from juice in comparison to smoothie. In vitro data with absorptive intestinal cells indicated that despite their weak chemical stability, ACN and 3,4-DHB could be detected at the basal side in their native forms. Whether smoothies as well as juices should be recommended to increase the intake of potentially health-promoting ACN and other polyphenols requires the consideration of other ingredients such as their relatively high sugar content.

High resolution mass spectrometry imaging of plant tissues: towards a plant metabolite atlas.

Mass spectrometry (MS) imaging provides spatial and molecular information for a wide range of compounds. This tool can be used to investigate metabolic changes in plant physiology and environmental interactions. A major challenge in our study was to prepare tissue sections that were compatible with high spatial resolution analysis and therefore dedicated sample preparation protocols were established and optimized for the physicochemical properties of all major plant organs. We combined high spatial resolution (5 µm), in order to detect cellular features, and high mass accuracy (<2 ppm root mean square error), for molecular specificity. Mass spectrometry imaging experiments were performed in positive and negative ion mode. Changes in metabolite patterns during plant development were investigated for germination of oilseed rape. The detailed localization of more than 90 compounds allowed assignment to metabolic processes and indicated possible functions in plant tissues. The 'untargeted' nature of MS imaging allows the detection of marker compounds for the physiological status, as demonstrated for plant-pathogen interactions. Our images show excellent correlation with optical/histological examination. In contrast to previous MS imaging studies of plants, we present a complete workflow that covers multiple species, such as oilseed rape, wheat seed and rice. In addition, different major plant organs and a wide variety of compound classes were analyzed. Thus, our method could be used to develop a plant metabolite atlas as a reference to investigate systemic and local effects of pathogen infection or environmental stress.

Metabolite localization by atmospheric pressure high-resolution scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging in whole-body sections and individual organs of the rove beetle Paederus riparius.

Mass spectrometry imaging provides for non-targeted, label-free chemical imaging. In this study, atmospheric pressure high-resolution scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) was used for the first time to describe the chemical distribution of the defensive compounds pederin, pseudopederin, and pederon in tissue sections (16 µm thick) of the rove beetle Paederus riparius. The whole-insect tissue section was scanned with a 20-µm step size. Mass resolution of the orbital trapping mass spectrometer was set to 100,000 at m/z 200. Additionally, organ-specific compounds were identified for brain, nerve cord, eggs, gut, ovaries, and malpighian tubules. To confirm the distribution of the specific compounds, individual organs from the insect were dissected, and MSI experiments were performed on the dissected organs. Three ganglia of the nerve cord, with a dimension of 250-500 µm, were measured with 10-µm spatial resolution. High-quality m/z images, based on high spatial resolution and high mass accuracy were generated. These features helped to assign mass spectral peaks with high confidence. Mass accuracy of the imaging experiments was <3 ppm root mean square error, and mapping of different compound classes from a single experiment was possible. This approach improved the understanding of the biochemistry of P. riparius. Concentration differences and distributions of pederin and its analogues could be visualized in the whole-insect section. Without any labeling, we assigned key lipids for specific organs to describe their location in the body and to identify morphological structures with a specificity higher than with staining or immunohistology methods.

Mass spectrometry imaging of biological tissue: an approach for multicenter studies.

Mass spectrometry imaging has become a popular tool for probing the chemical complexity of biological surfaces. This led to the development of a wide range of instrumentation and preparation protocols. It is thus desirable to evaluate and compare the data output from different methodologies and mass spectrometers. Here, we present an approach for the comparison of mass spectrometry imaging data from different laboratories (often referred to as multicenter studies). This is exemplified by the analysis of mouse brain sections in five laboratories in Europe and the USA. The instrumentation includes matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF), MALDI-QTOF, MALDI-Fourier transform ion cyclotron resonance (FTICR), atmospheric-pressure (AP)-MALDI-Orbitrap, and cluster TOF-secondary ion mass spectrometry (SIMS). Experimental parameters such as measurement speed, imaging bin width, and mass spectrometric parameters are discussed. All datasets were converted to the standard data format imzML and displayed in a common open-source software with identical parameters for visualization, which facilitates direct comparison of MS images. The imzML conversion also allowed exchange of fully functional MS imaging datasets between the different laboratories. The experiments ranged from overview measurements of the full mouse brain to detailed analysis of smaller features (depending on spatial resolution settings), but common histological features such as the corpus callosum were visible in all measurements. High spatial resolution measurements of AP-MALDI-Orbitrap and TOF-SIMS showed comparable structures in the low-micrometer range. We discuss general considerations for planning and performing multicenter studies in mass spectrometry imaging. This includes details on the selection, distribution, and preparation of tissue samples as well as on data handling. Such multicenter studies in combination with ongoing activities for reporting guidelines, a common data format (imzML) and a public data repository can contribute to more reliability and transparency of MS imaging studies.

A comprehensive high-resolution mass spectrometry approach for characterization of metabolites by combination of ambient ionization, chromatography and imaging methods.

RATIONALE: An ideal method for bioanalytical applications would deliver spatially resolved quantitative information in real time and without sample preparation. In reality these requirements can typically not be met by a single analytical technique. Therefore, we combine different mass spectrometry approaches: chromatographic separation, ambient ionization and imaging techniques, in order to obtain comprehensive information about metabolites in complex biological samples. METHODS: Samples were analyzed by laser desorption followed by electrospray ionization (LD-ESI) as an ambient ionization technique, by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging for spatial distribution analysis and by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) for quantitation and validation of compound identification. All MS data were acquired with high mass resolution and accurate mass (using orbital trapping and ion cyclotron resonance mass spectrometers). Grape berries were analyzed and evaluated in detail, whereas wheat seeds and mouse brain tissue were analyzed in proof-of-concept experiments. RESULTS: In situ measurements by LD-ESI without any sample preparation allowed for fast screening of plant metabolites on the grape surface. MALDI imaging of grape cross sections at 20 µm pixel size revealed the detailed distribution of metabolites which were in accordance with their biological function. HPLC/ESI-MS was used to quantify 13 anthocyanin species as well as to separate and identify isomeric compounds. A total of 41 metabolites (amino acids, carbohydrates, anthocyanins) were identified with all three approaches. Mass accuracy for all MS measurements was better than 2 ppm (root mean square error). CONCLUSIONS: The combined approach provides fast screening capabilities, spatial distribution information and the possibility to quantify metabolites. Accurate mass measurements proved to be critical in order to reliably combine data from different MS techniques. Initial results on the mycotoxin deoxynivalenol (DON) in wheat seed and phospholipids in mouse brain as a model for mammalian tissue indicate a broad applicability of the presented workflow.

Analysis of cyathane-type diterpenoids from Cyathus striatus and Hericium erinaceus by high-resolution MALDI MS imaging.

Fungal secondary metabolites in both fruiting bodies and pellets from submerged cultures of basidiomycetes were analyzed by atmospheric pressure matrix-assisted laser desorption/ionization-mass spectrometry imaging at a lateral resolution of 15 µm, a mass resolution of 140,000 at m/z 200 and a mass accuracy of better than 2 ppm. The striatals A, B, C, and D, and a number of erinacine type metabolites were detected in the basidiomycetes Cyathus striatus and Hericium erinaceus, respectively. The two fungi were selected as model species, as they are well-known for efficient production of terpenoid secondary metabolites with interesting biological activities, e.g., antibacterial, fungicidal, cytotoxic properties, and stimulating effects on nerve growth factor synthesis. The localization of metabolites revealed a mostly homogeneous distribution of the striatals in the pellets of C. striatus, while a concentration gradient, increasing to the center, was observed in the pellets of H. erinaceus. A mostly homogeneous distribution of metabolites was also found in the fruiting body of H. erinaceus.

Proteomics study of silver nanoparticles toxicity on Bacillus thuringiensis.

Emerging technologies in functional genomics and proteomics provide a way of achieving high-throughput analyses, understanding effects on protein populations and sub-populations and follow up environmental stresses. To accomplish these, the action of homemade spherical Silver nanoparticles colloidal suspension (AgNPs) against Bacillus thuringiensis (isolate from Oryza sativa L. rhizosphere) was investigated by a proteomic approach (2-DE and NanoLC/FT-ICR MS identification). Thirty four responsive (up/down regulated) proteins were identified. Proteomic results revealed that an exposure of B. thuringiensis cells with different concentrations of AgNPs resulted in an accumulation of envelope protein precursors, indicative of the dissipation of a proton motive force. Identified proteins are involved in oxidative stress tolerance, metal detoxification, transcription and elongation processes, protein degradation, cytoskeleton remodeling and cell division. The expression pattern of these proteins and their possible involvement in the nontoxicity mechanisms were discussed.

Proteomics study of silver nanoparticles toxicity on Oryza sativa L.

The increasing use of silver nanoparticles, (AgNPs), will inevitably result in their release into the environment and thereby cause the exposure to plants. It was claimed that using AgNPs is a safe and efficient method to preserve and treat agents of disease in agriculture. This study tries to understand the protein populations and sub-populations and follow up environmental AgNPs stresses. To accomplish these, the action of homemade spherical AgNPs colloidal suspension against Oryza sativa L. was investigated by a proteomic approach (2-DE and NanoLC/FT-ICR MS identification). Twenty-eight responsive (decrement/increment in abundance) proteins were identified. Proteomic results revealed that an exposure of O. sativa L., root with different concentrations of AgNPs resulted in an accumulation of protein precursors, indicative of the dissipation of a proton motive force. The identified proteins are involved in oxidative stress tolerance, Ca(2+) regulation and signaling, transcription and protein degradation, cell wall and DNA/RNA/protein direct damage, cell division and apoptosis. The expression pattern of these proteins and their possible involvement in the nontoxicity mechanisms were discussed.