Desorption electrospray ionization then MALDI mass spectrometry imaging of lipid and protein distributions in single tissue sections

Imaging mass spectrometry (MS) is a powerful technique for mapping the spatial distributions of a wide range of chemical compounds simultaneously from a tissue section. Co-localization of the distribution of individual molecular species, including particular lipids and proteins, and correlation with the morphological features of a single tissue section are highly desirable for comprehensive tissue analysis and disease diagnosis. We now report on the use, in turn, of desorption electrospray ionization (DESI), matrix assisted laser desorption ionization (MALDI), and then optical microscopy to image lipid and protein distributions in a single tissue section. This is possible through the use of histologically compatible DESI solvent systems, which allow for sequential analyses of the same section by DESI then MALDI. Hematoxylin and eosin (H&E) staining was performed on the same section after removal of the MALDI matrix. This workflow allowed chemical information to be unambiguously matched to histological features in mouse brain tissue sections. The lipid sulfatide (24:1), detected at m/z 888.8 by DESI imaging, was colocalized with the protein MBP isoform 8, detected at m/z 14117 by MALDI imaging, in regions corresponding to the corpus callosum substructure of the mouse brain, as confirmed in the H&E images. Correlation of lipid and protein distributions with histopathological features was also achieved for human brain cancer samples. Higher tumor cell density was observed in regions demonstrating higher relative abundances of oleic acid, detected by DESI imaging at m/z 281.4, and the protein calcyclin, detected by MALDI at m/z 10085, for a human glioma sample. Since correlation between molecular signatures and disease state can be achieved, we expect that this methodology will significantly enhance the value of MS imaging in molecular pathology for diagnosis.     

Sensitive detection of Bacillus anthracis spores by immunocapture and liquid chromatography-tandem mass spectrometry

Bacillus anthracis is one of the most dangerous agents of the bioterrorism threat. We present here a sensitive immuno-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) approach to spore detection in complex environmental samples. It is based on the combined specificity and sensitivity of two techniques: immunocapture and targeted mass spectrometry. The immunocapture step, realized directly on the intact spores, is essential for their selective isolation and concentration from complex environmental samples. After parallel trypsin and Glu-C digestions, proteotypic peptides corresponding to small acid-soluble spore protein-B (SASP-B) are specifically monitored in the multiple reaction monitoring (MRM) mass spectrometry mode. Peptide ratio is carefully monitored and provides an additional level of specificity, which is shown to be highly useful for distinguishing closely related samples and avoiding false-positive/negative results. Sensitivity at the level of the infectious dose is demonstrated, with limits of detection of 7 × 10(3) spores/mL of milk or 10 mg of soil. This mass spectrometry approach is thus complementary to polymerase chain reaction (PCR) techniques.     

Analysis of a polydisperse polyrotaxane based on poly(ethylene oxide) and α-cyclodextrins using nanoelectrospray and LTQ-Orbitrap

Polyrotaxanes (PR) are among the most studied interlocked molecules in the field of supramolecular chemistry. Cyclodextrin based polyrotaxanes (CD based PRs) are well-known to be difficult to analyze by mass spectrometry (MS). Nanoelectrospray (nanoESI) employed during mass spectrometry (MS) and tandem mass spectrometry (MS/MS) experiments turns out to be particularly useful to analyze these noncovalent assemblies. While ESI/nanoESI based spectra usually contain multicharged species which greatly complicate the interpretation, particularly for such complex mixtures analysis, the hyphenation with a high resolution analyzer such as Orbitrap could overcome this limitation. This Article reports efforts to achieve a detailed structural deciphering by nanoESI-MS and nanoESI-MS/MS of CD based PRs constituted of αCDs, unmodified or surrounded by 1 or 2 sulfation(s), which were threaded along polydisperse poly(ethylene oxide) α,ω-dipyrenyl chains. The described method is more sensitive and less sample consuming than a typical NMR experiment and in good agreement with size-exclusion chromatography (SEC) results. Moreover, as compared to MALDI-TOF MS analysis, all populations were presumably elucidated without discrimination effect. Therefore, this MS development allowed us to estimate the PR sample content with 16 to 35 ethylene oxide units, 1 to 5 αCDs threaded, and 0 to 10 sulfo groups grafted on the overall CDs. Finally, the method afforded the possibility to unambiguously attribute supramolecular architectures from 2276.0278 to 7767.8342 g·mol(-1) corresponding to poly[2]- to poly[6]rotaxanes.     

Paraquat-loaded alginate/chitosan nanoparticles: preparation, characterization and soil sorption studies

Agrochemicals are amongst the contaminants most widely encountered in surface and subterranean hydrological systems. They comprise a variety of molecules, with properties that confer differing degrees of persistence and mobility in the environment, as well as different toxic, carcinogenic, mutagenic and teratogenic potentials, which can affect non-target organisms including man. In this work, alginate/chitosan nanoparticles were prepared as a carrier system for the herbicide paraquat. The preparation and physico-chemical characterization of the nanoparticles was followed by evaluation of zeta potential, pH, size and polydispersion. The techniques employed included transmission electron microscopy, differential scanning calorimetry and Fourier transform infrared spectroscopy. The formulation presented a size distribution of 635 ± 12 nm, polydispersion of 0.518, zeta potential of -22.8 ± 2.3 mV and association efficiency of 74.2%. There were significant differences between the release profiles of free paraquat and the herbicide associated with the alginate/chitosan nanoparticles. Tests showed that soil sorption of paraquat, either free or associated with the nanoparticles, was dependent on the quantity of organic matter present. The results presented in this work show that association of paraquat with alginate/chitosan nanoparticles alters the release profile of the herbicide, as well as its interaction with the soil, indicating that this system could be an effective means of reducing negative impacts caused by paraquat.     

Method for the direct observation and quantification of survival of bacteria attached to negatively or positively charged surfaces in an aqueous medium

The risk of groundwater contamination by microbial pathogens is linked to their survival in the subsurface. Although there is a large body of literature on the inactivation behavior of suspended (planktonic) microorganisms, little is known about the inactivation of bacteria when attached to sand grain surfaces in groundwater aquifers. The main goal of this study was to develop a fluorescence-based experimental technique for evaluating the extent of inactivation over time of bacteria adhered onto a surface in an aqueous environment. Key features of the developed technique are as follows: (i) attached cells do not need to be removed from the surface of interest for quantification, (ii) bacterial inactivation can be examined in real-time for prolonged time periods, and (iii) the system remains undisturbed (i.e., the aqueous environment is unchanged) during the assay. A negatively or positively charged substrate (i.e., bare or coated glass slide) was mounted in a parallel-plate flow cell, bacteria were allowed to attach onto the substrate, and the loss of bacterial membrane integrity and respiratory activity were investigated as a function of time by fluorescence microscopy using Live/Dead BacLight and BacLight RedoxSensor CTC (5-cyano-2,3-ditolyl tetrazolium chloride) viability assays. These two different measures of bacterial inactivation result in comparable trends in bacterial inactivation, confirming the validity of the experimental technique. The results of this work show that the developed technique is sensitive enough to distinguish between the inactivation kinetics of different representative bacteria attached to either a negatively charged (bare glass) surface or a positively charged (coated glass) surface. Hence, the technique can be used to characterize bacterial inactivation kinetics when attached to environmentally relevant surfaces over a broad range of groundwater chemistries.     

Dissolution of technetium(IV) oxide by natural and synthetic organic ligands under both reducing and oxidizing conditions

Technetium-99 (Tc) in nuclear waste is a significant environmental concern due to its long half-life and high mobility in the subsurface. Reductive precipitation of technetium(IV) oxides [TcO(2)(s)] is an effective means of immobilizing Tc, thereby impeding its migration in groundwater. However, technetium(IV) oxides are subject to dissolution by oxidants and/or complexing agents. In this study we ascertain the effects of a synthetic organic ligand, ethylenediaminetetraacetate (EDTA), and two natural humic isolates on the dissolution and solubility of technetium(IV) oxides. Pure synthetic technetium(IV) oxide (0.23 mM) was used in batch experiments to determine dissolution kinetics at pH ～6 under both reducing and oxidizing conditions. All organic ligands were found to enhance the dissolution of technetium(IV) oxides, increasing their solubility from ～10(-8) M (without ligands) to 4 × 10(-7) M under strictly anoxic conditions. Reduced Tc(IV) was also found to reoxidize rapidly under oxic conditions, with an observed oxidative dissolution rate approximately an order of magnitude higher than that of ligand-promoted dissolution under reducing conditions. Significantly, oxidative dissolution was inhibited by EDTA but enhanced by humic acid compared to experiments without any complexing agents. The redox functional properties of humics, capable of facilitating intramolecular electron transfer, may account for this increased oxidation rate under oxic conditions. Our results highlight the importance of complex interactions for the stability and mobility of Tc and thus for the long-term fate of Tc in contaminated environments.     

Interactions of Tc(IV) with humic substances

To understand the key processes affecting 99Tc mobility in the subsurface and help with the remediation of contaminated sites, the binding constants of several humic substances (humic and fulvic acids) with Tc(IV) were determined, using a solvent extraction technique. The novelty of this paper lies in the determination of the binding constants of the complexes formed with the individual species TcO(OH)+ and TcO(OH)2(0). Binding constants were found to be 6.8 and between 3.9 and 4.3, for logβ1,-1,1 and logβ1,-2,1, respectively; these values were little modified by a change of ionic strength, in most cases, between 0.1 and 1.0 M, nor were they by the nature and origin of the humic substances. Modeling calculations based on these show TcO(OH)-HA to be the predominant complex in a system containing 20 ppm HA and in the 4-6 pH range, whereas TcO(OH)2(0) and TcO(OH)2-HA are the major species, in the pH 6-8 range.     

Texture and surface morphology effects on tetragonal phase stabilization in ZrO2 films deposited by metal-organic chemical vapor deposition

Tetragonal zirconia coatings (ZrO2) without doping any trivalent impurities have been deposited by metal-organic chemical vapor deposition (MOCVD) on (100) Si single crystals, using Zr(thd)4 precursor. The surface and cross-section morphologies were observed with Field-Emission-Gun Scanning Electron Microscopy (FEG-SEM). The crystalline structures were characterized by grazing incident X-ray diffraction (GIXRD). Crystallographic textures of these films were studied for both {011}t and {110}t planes by pole figure recording by X-ray diffraction under a 4-circle goniometer. The internal stresses were measured with the use of sin2 psi method. In order to study the relationship of microstructures and tetragonal phase stabilization in ZrO2 films, annealing experiments were taken at different temperature. The results show that the critical crystallite size for tetragonal to monoclinic phase transformation is different for samples with different initial microstructures. Besides the critical crystallite size and the residual stress, the texture and crystallite morphology of the ZrO2 films are responsible for the stabilization of the metastable tetragonal phase.