Land-use and fire drive temporal patterns of soil solution chemistry and nutrient fluxes.

Land-use type and ecosystem disturbances are important drivers for element cycling and bear the potential to modulate soil processes and hence ecosystem functions. To better understand the effect of such drivers on the magnitude and temporal patterns of organic matter (OM) and associated nutrient fluxes in soils, continuous flux monitoring is indispensable but insufficiently studied yet. We conducted a field study to elucidate the impact of land-use and surface fires on OM and nutrient fluxes with soil solution regarding seasonal and temporal patterns analyzing short (<3months) and medium-term (3-12months) effects. Control and prescribed fire-treated topsoil horizons in beech forests and pastures were monitored biweekly for dissolved and particulate OM (DOM, POM) and solution chemistry (pH value, elements: Ca, Mg, Na, K, Al, Fe, Mn, P, S, Si) over one post-fire year. Linear mixed model analyses exhibited that mean annual DOM and POM fluxes did not differ between the two land-use types, but were subjected to strong seasonal patterns. Fire disturbance significantly lowered the annual soil solution pH in both land-uses and increased water fluxes, while DOC fluxes remained unaffected. A positive response of POC and S to fire was limited to short-term effects, while amplified particulate and dissolved nitrogen fluxes were observed in the longer run and co-ocurred with accelerated Ca and Mg fluxes. In summary, surface fires generated stronger effects on element fluxes than the land-use. Fire-induced increases in POM fluxes suggest that the particulate fraction represent a major pathway of OM translocation into the subsoil and beyond. With regard to ecosystem functions, pasture ecosystems were less prone to the risk of nutrient losses following fire events than the forest. In pastures, fire-induced base cation export may accelerate soil acidification, consequently exhausting soil buffer systems and thus may reduce the resilience to acidic depositions and disturbances.

In situ monitoring of molecular changes during cell differentiation processes in marine macroalgae through mass spectrometric imaging.

Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) was employed to discriminate between cell differentiation processes in macroalgae. One of the key developmental processes in the algal life cycle is the production of germ cells (gametes and zoids). The gametogenesis of the marine green macroalga Ulva mutabilis (Chlorophyta) was monitored by metabolomic snapshots of the surface, when blade cells differentiate synchronously into gametangia and giving rise to gametes. To establish MSI for macroalgae, dimethylsulfoniopropionate (DMSP), a known algal osmolyte, was determined. MSI of the surface of U. mutabilis followed by chemometric data analysis revealed dynamic metabolomic changes during cell differentiation. DMSP and a total of 55 specific molecular biomarkers, which could be assigned to important stages of the gametogenesis, were detected. Our research contributes to the understanding of molecular mechanisms underlying macroalgal cell differentiation. Graphical abstract Molecular changes during cell differentiation of the marine macroalga Ulva were visualized by matrix assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI).

Spatial and Temporal Localization of Flavonoid Metabolites in Strawberry Fruit (Fragaria × ananassa).

Flavonoids are important metabolites in strawberries (Fragaria × ananassa) because they accomplish an extensive collection of physiological functions and are valuable for human health. However, their localization within the fruit tissue has not been extensively explored. Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) was employed to shed light on the spatial distribution of flavonoids during fruit development. One wild-type (WT) and two transgenic lines were compared, wherein the transgenic enzymes anthocyanidin reductase (ANRi) and flavonol synthase (FLSi), respectively, were down-regulated using an RNAi-based silencing approach. In most cases, fruit development led to a reduction of the investigated flavonoids in the fruit tissue; as a consequence, they were exclusively present in the skin of mature red fruits. In the case of (epi)catechin dimer, both the ANRi and the WT phenotypes revealed low levels in mature red fruits, whereas the ANRi line bore the lowest relative concentration, as analyzed by liquid chromatography-electrospray ionization multiple-step mass spectrometry (LC-ESI-MSn).

Tracing the fate and transport of secondary plant metabolites in a laboratory mesocosm experiment by employing mass spectrometric imaging.

Mass spectrometric imaging (MSI) has received considerable attention in recent years, since it allows the molecular mapping of various compound classes, such as proteins, peptides, glycans, secondary metabolites, lipids, and drugs in animal, human, or plant tissue sections. In the present study, the application of laser-based MSI analysis of secondary plant metabolites to monitor their transport from the grass leaves of Dactylis glomerata, over the crop of the grasshopper Chorthippus dorsatus to its excrements, and finally in the soil solution is described. This plant-herbivore-soil pathway was investigated under controlled conditions by using laboratory mesocosms. From six targeted secondary plant metabolites (dehydroquinic acid, quinic acid, apigenin, luteolin, tricin, and rosmarinic acid), only quinic acid, and dehydroquinic acid, an in-source-decay (ISD) product of quinic acid, could be traced in nearly all compartments. The tentative identification of secondary plant metabolites was performed by MS/MS analysis of methanol extracts prepared from the investigated compartments, in both the positive and negative ion mode, and subsequently compared with the results generated from the reference standards. Except for tricin, all secondary metabolites could be tentatively identified by this approach. Additional liquid-chromatography mass spectrometry (LC-MS) experiments were carried out to verify the MSI results and revealed the presence of quinic acid only in grass and chewed grass, whereas apigenin-hexoside-pentoside and luteolin-hexoisde-pentoside could be traced in the grasshopper body and excrement extracts. In summary, the MSI technique shows a trade-off between sensitivity and spatial resolution. Graphical abstract Monitoring quinic acid in a mesocosm experiment by mass spectrometric imaging (MSI).

Effect of ecosystem type and fire on chemistry of WEOM as measured by LDI-TOF-MS and NMR.

Soil organic matter (SOM) and its water-soluble components play an important role in terrestrial carbon cycling and associated ecosystem functions. Chemically, they are complex mixtures of organic compounds derived from decomposing plant material, microbial residues, as well as root exudates, and soil biota. To test the effect of the ecosystem type (forest and grassland) and fires events on the chemistry of dissolved organic matter (DOM), we applied a combination of laser-desorption/ionization time-of-flight mass spectrometry (LDI-TOF-MS) and 2D (1H and 13C) nuclear magnetic resonance (NMR) spectroscopy to water-extractable organic matter (WEOM) from a range of top soil samples. The aim was to assess the suitability of LDI-TOF-MS for the rapid characterization of WEOM. Therefore, we evaluated the effects of sample (pH and dilution) conditions and use of positive or negative reflector mode to identify the conditions under which LDI-TOF-MS best distinguished between WEOM from different sources. Thirty-six samples were measured with both analytical techniques and their chemical patterns were statistically evaluated to distinguish firstly the effect of the type of ecosystem (forest versus grassland) on WEOM characteristics, and secondly the impact of fire on the chemical composition of WEOM. The nonmetric multidimensional scaling (NMDS) analysis of the most suitable experimental LDI-TOF-MS conditions showed a clear separation between the type of vegetation and fire-induced changes, mostly reflecting the presence of poly(ethylene glycol) in grassland soils. Discrimination among WEOM from different vegetation types was preserved in the fire treated samples. The calculation of the relative abundance of certain functional structures in the WEOM samples revealed a common composition of forest and grassland WEOM, with polysaccharides and proteins making up to 60%. The compositional impact of forest fire on WEOM was more pronounced compared to the one of grassland, leading to a decline in the main components, and an increase in amino-sugars, fatty acids, and sterols. The recorded 1H NMR and heteronuclear single quantum coherence (HSQC) spectra showed a decrease of the carbohydrate signal in WEOM from fire-treated samples, which was more pronounced in forest than in grassland soils.

Spatial segmentation of MALDI FT-ICR MSI data: a powerful tool to explore the head and neck tumor in situ lipidome.

Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI MSI) is a well-established analytical technique for determining spatial localization of lipids in biological samples. The use of Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometers for the molecular imaging of endogenous compounds is gaining popularity, since the high mass accuracy and high mass resolving power enables accurate determination of exact masses and, consequently, a more confident identification of these molecules. The high mass resolution FT-ICR imaging datasets are typically large in size. In order to analyze them in an appropriate timeframe, the following approach has been employed: the FT-ICR imaging datasets were spatially segmented by clustering all spectra by their similarity. The resulted spatial segmentation maps were compared with the histologic annotation. This approach facilitates interpretation of the full datasets by providing spatial regions of interest. The application of this approach, which has originally been developed for MALDI-TOF MSI datasets, to the lipidomic analysis of head and neck tumor tissue revealed new insights into the metabolic organization of the carcinoma tissue.

Phenalenone-type phytoalexins mediate resistance of banana plants (Musa spp.) to the burrowing nematode Radopholus similis.

The global yield of bananas-one of the most important food crops-is severely hampered by parasites, such as nematodes, which cause yield losses up to 75%. Plant-nematode interactions of two banana cultivars differing in susceptibility to Radopholus similis were investigated by combining the conventional and spatially resolved analytical techniques (1)H NMR spectroscopy, matrix-free UV-laser desorption/ionization mass spectrometric imaging, and Raman microspectroscopy. This innovative combination of analytical techniques was applied to isolate, identify, and locate the banana-specific type of phytoalexins, phenylphenalenones, in the R. similis-caused lesions of the plants. The striking antinematode activity of the phenylphenalenone anigorufone, its ingestion by the nematode, and its subsequent localization in lipid droplets within the nematode is reported. The importance of varying local concentrations of these specialized metabolites in infected plant tissues, their involvement in the plant's defense system, and derived strategies for improving banana resistance are highlighted.

Mass spectrometric imaging of synthetic polymers.

The analysis of synthetic polymers represents today an important part of polymer science to determine their physical properties and to optimize the performance of polymeric materials for block copolymers as well as blend systems. The characterization can easily and rapidly be performed by mass spectrometry. In particular, the film formation of a synthetic polymer is of interest in material research and quality control, which can be determined by employing mass spectrometric imaging (MSI) using secondary ion mass spectrometry (SIMS) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. MALDI-MSI has been rapidly improved for the analysis of tissue cross-sections due to its soft ionization and accessible m/z range, which both also play an important role in polymer science. On the other hand, SIMS-MSI enables a sub-micrometer molecular spatial resolution, which is limited in MALDI-MSI due to the spatial resolution capabilities of the laser desorption process. The aim of the present contribution is to summarize recent advances in both imaging techniques for the analysis of synthetic polymers and to highlight their capabilities to correlate several imaging modalities in future applications.

4-Deoxyaurone formation in Bidens ferulifolia (Jacq.) DC.

The formation of 4-deoxyaurones, which serve as UV nectar guides in Bidens ferulifolia (Jacq.) DC., was established by combination of UV photography, mass spectrometry, and biochemical assays and the key step in aurone formation was studied. The yellow flowering ornamental plant accumulates deoxy type anthochlor pigments (6'-deoxychalcones and the corresponding 4-deoxyaurones) in the basal part of the flower surface whilst the apex contains only yellow carotenoids. For UV sensitive pollinating insects, this appears as a bicoloured floral pattern which can be visualized in situ by specific ammonia staining of the anthochlor pigments. The petal back side, in contrast, shows a faintly UV absorbing centre and UV absorbing rays along the otherwise UV reflecting petal apex. Matrix-free UV laser desorption/ionisation mass spectrometric imaging (LDI-MSI) indicated the presence of 9 anthochlors in the UV absorbing areas. The prevalent pigments were derivatives of okanin and maritimetin. Enzyme preparations from flowers, leaves, stems and roots of B. ferulifolia and from plants, which do not accumulate aurones e.g. Arabidopsis thaliana, were able to convert chalcones to aurones. Thus, aurone formation could be catalyzed by a widespread enzyme and seems to depend mainly on a specific biochemical background, which favours the formation of aurones at the expense of flavonoids. In contrast to 4-hydroxyaurone formation, hydroxylation and oxidative cyclization to the 4-deoxyaurones does not occur in one single step but is catalyzed by two separate enzymes, chalcone 3-hydroxylase and aurone synthase (catechol oxidase reaction). Aurone formation shows an optimum at pH 7.5 or above, which is another striking contrast to 4-hydroxyaurone formation in Antirrhinum majus L. This is the first example of a plant catechol oxidase type enzyme being involved in the flavonoid pathway and in an anabolic reaction in general.

Application of matrix-assisted laser desorption/ionization mass spectrometric imaging for photolithographic structuring.

The aim of this contribution is the application of matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) in the area of photolithographic structuring. As proof of concept, this method was used to image an UV exposed negative photoresist layer, which is generally used to manufacture printed circuit boards (PCB) for electronic components. The negative photoresist layer consisting of the main component novolac, benzophenone as the active component, and the solvent tetrahydrofuran was mixed with the matrix dithranol and the salt additive LiTFA and spin-coated onto an ITO-conductive glass slide. To imprint an image on the created surface, a transparency with a printed wiring diagram was placed on top of it and irradiated by UV light for 15 min. The inspection of the efficient imprinting of the microstructure onto the photoresist layer was performed by MALDI-MSI. This unique application represents a further step toward the surface analysis of polymer films by this emerging life science imaging technique.

Determination of the relative ligand-binding strengths in heteroleptic Ir(III) complexes by ESI-Q-TOF tandem mass spectrometry.

An electrospray ionization quadrupole time-of-flight mass spectrometer has been utilized to investigate the relative ligand-binding strengths in a series of heteroleptic-charged iridium(III) complexes of the general formula [(C^N)(2) Ir(III) (S-tpy)](PF(6) ) by using variable collision energies. Collision-induced dissociation experiments were performed in order to study the stability of the Ir(III) complexes that are, for instance, suitable phosphors in light-emitting electrochemical cells. The ratio of signal intensities belonging to the fragment and the undissociated complex depends on the collision energy applied for the tandem mass spectra (MS/MS) analysis. By defining the threshold collision energy and the point of complete complex dissociation, it is possible to estimate the relative complex stabilities depending on the nature of the coordinated ligands [i.e. type of cyclometalating ligand (C^N), substituents on the S-shaped terpyridine (S-tpy)]. The collision energy values differed as a function of the coordination sphere of the Ir(III) centers.

Tandem mass spectrometry of poly(ethylene imine)s by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI).

In this contribution, linear poly(ethylene imine) (PEI) polymers, which are of importance in gene delivery, are investigated in detail by using electrospray ionization-quadrupole-time of flight (ESI-Q-TOF) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS). The analyzed PEIs with different end groups were synthesized using the polymerization of substituted 2-oxazoline via a living cationic ring-opening polymerization (CROP) and a subsequent hydrolysis under acidic conditions. The main goal of this study was to identify linear PEI polymers in a detailed way to gain information about their fragmentation pathways. For this purpose, a detailed characterization of three different linear PEIs was performed by using ESI-Q-TOF and MALDI-TOF MS in combination with collision-induced dissociation (CID) experiments. In ESI-MS as well as MALDI-MS analysis, the obtained spectra of PEIs resulted in fitting mass distributions for the investigated PEIs. In the tandem MS analysis, a 1,2-hydride shift with a charge-remote rearrangement via a four-membered cyclic transition state, as well as charge-induced fragmentation reactions, was proposed as the main fragmentation mechanisms according to the obtained fragmentation products from the protonated parent peaks. In addition, heterolytic and homolytic cleavages were proposed as alternative fragmentation pathways. Moreover, a 1,4-hydrogen elimination was proposed to explain different fragmentation products obtained from the sodiated parent peaks.

Application of matrix-assisted laser desorption/ionization mass spectrometric imaging to monitor surface changes of UV-irradiated poly(styrene) films.

This study presents the application of matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) to monitor changes occurring at polymer surfaces. As an example, a poly(styrene) (PS) film was irradiated with ultraviolet (UV) light at 254 nm for different time intervals, while areas of the film were protected from UV light by covering it with an aluminum mask. After the UV treatment, the polymer surface was analyzed by MALDI-MSI. Time-dependent photo-induced cross-linking of the polymer film was observed, and a correlation curve between UV radiation time and area of cross-linking was constructed. This represents the first step towards the surface analysis of polymer components of photoresists and top coatings of cars, and it will also enable a new characterization strategy for combinatorial material research.

Analysis of different synthetic homopolymers by the use of a new calculation software for tandem mass spectra.

The manual interpretation of tandem mass spectra of synthetic polymers is very time-consuming. Therefore, a new software tool was developed to accelerate the interpretation of spectra obtained without requiring any further knowledge about the polymer class or the fragmentation behavior under high-energy collision-induced dissociation (CID) conditions. The software only requires an alphabetical list of elements and a peak list of the measured substance as an xml file for the evaluation of the chosen mass spectrum. Tandem mass spectra of different homopolymers, like poly(2-oxazoline)s, poly(ethylene glycol) and poly(styrene), were interpreted by the new software tool. This contribution describes a fast and automated software tool for the rapid analysis of homopolymers.

Combinatorial optimization of multiple MALDI matrices on a single tissue sample using inkjet printing.

Taking advantage of the drop-on-demand capabilities of inkjet printing, the first example of a single tissue being used as a substrate for preparing combinatorial arrays of different matrix-assisted laser desorption/ionization (MALDI) matrices in multiple concentrations on a single chip is reported. By varying the number of droplets per spot that were printed, a gradient array of different amounts of matrix material could be printed on a single chip, while the selection of matrices could be adjusted by switching different matrix materials. The result was a two-dimensional array of multiple matrices on a single tissue slice, which could be analyzed microscopically and by MALDI to elucidate which combination of matrix and printing conditions offered the best resolution in terms of spot-to-spot distance and signal-to-noise ratios for proteins in the recorded MS spectra. This combinatorial approach enables the efficient optimization of possible matrices in an organized, side-by-side array format, which can particularly be useful for the detection of specific protein markers.

Depicting the spatial distribution of proteins in human tumor tissue combining SELDI and MALDI imaging and immunohistochemistry.

Carcinoma tissue consists of not only tumor cells but also fibroblasts, endothelial cells or vascular structures, and inflammatory cells forming the supportive tumor stroma. Therefore, the spatial distribution of proteins that promote growth and proliferation in these complex functional units is of high interest. Matrix-assisted laser desorption/ionization imaging mass spectrometry is a newly developed technique that generates spatially resolved profiles of protein signals directly from thin tissue sections. Surface-enhanced laser desorption/ionization mass spectrometry (MS)combined with tissue microdissection allows analysis of defined parts of the tissue with a higher sensitivity and a broader mass range. Nevertheless, both MS-based techniques have a limited spatial resolution. IHC is a technique that allows a resolution down to the subcellular level. However, the detection and measurement of a specific protein expression level is possible only by semiquantitative methods. Moreover, prior knowledge about the identity of the proteins of interest is necessary. In this study, we combined all three techniques to gain highest spatial resolution, sensitivity, and quantitative information. We used frozen tissue from head and neck tumors and chose two exemplary proteins (HNP1-3 and S100A8) to highlight the advantages and disadvantages of each technique. It could be shown that the combination of these three techniques results in congruent but also synergetic data.

Tandem mass spectrometry of synthetic polymers.

The detailed characterization of macromolecules plays an important role for synthetic chemists to define and specify the structure and properties of the successfully synthesized polymers. The search for new characterization techniques for polymers is essential for the continuation of the development of improved synthesis methods. The application of tandem mass spectrometry for the detailed characterization of synthetic polymers using the soft ionization techniques matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS), which became the basic tools in proteomics, has greatly been increased in recent years and is summarized in this perspective. Examples of a variety of homopolymers, such as poly(methyl methacrylate), poly(ethylene glycol), as well as copolymers, e.g. copolyesters, are given. The advanced mass spectrometric techniques described in this review will presumably become one of the basic tools in polymer chemistry in the near future.

The brain proteome profile is highly conserved between Prnp-/- and Prnp+/+ mice.

The aim of this study is to compare the proteome of Prnp-/- (Zürich I) gene-ablated mouse brains with wild-type mouse brains. Fluorescence two-dimensional-difference gel electrophoresis (DIGE) and isotope-coded protein labeling (ICPL) were applied for brain homogenates. Similar quantitative protein profiles (

Analysis of the HUPO Brain Proteome reference samples using 2-D DIGE and 2-D LC-MS/MS.

Within the Human Proteome Organization (HUPO) Brain Proteome Project, a pilot study was launched with reference samples shipped to nine international laboratories (see Hamacher et al., this Special Issue) to evaluate different proteome approaches in neuroscience and to build up a first version of a brain protein database. One part of the study addresses quantitative proteome alterations between three developmental stages (embryonic day 16; postnatal day 7; 8 weeks) of mouse brains. Five brains per stage were differentially analyzed by 2-D DIGE using internal standardization and overlapping pH gradients (pH 4-7 and 6-9). In total, 214 protein spots showing stage-dependent intensity alterations (> two-fold) were detected, 56 of which were identified. Several of them, e.g. members of the dihydropyrimidinase family, are known to be associated with brain development. To feed the HUPO BPP brain protein database, a robust 2-D LC-MS/MS method was applied to murine postnatal day 7 and human post-mortem brain samples. Using MASCOT and the IPI database, 350 human and 481 mouse proteins could be identified by at least two different peptides. The data are accessible through the PRIDE database (http://www.ebi.ac.uk/pride/).