A forefront of the research on Alzheimer's disease (AD) is the interaction of amyloid beta (Abeta) peptides with redox metal ions (e.g., Cu(II), Fe(III), and Fe(II)) and the biological relevance of ...the Abeta-metal complexes to neuronal cell loss and homeostasis of essential metals and other cellular species. This work is concerned with the kinetic and mechanistic studies of the ascorbic acid oxidation reaction by molecular oxygen that is facilitated by Cu(II) complexes with Abeta(1-16), Abeta(1-42), and aggregates of Abeta(1-42). The reaction rate was found to linearly increase with the concentrations of Abeta-Cu(II) and dissolved oxygen and be invariant with high ascorbic acid concentrations. The rate constants were measured to be 117.2 +/- 15.4 and 15.8 +/- 2.8 M(-1) s(-1) at low (<100 muM) and high AA concentrations, respectively. Unlike free Cu(II), in the presence of AA, Abeta-Cu(II) complexes facilitate the reduction of oxygen by producing H(2)O(2) as a major product. Such a conclusion is drawn on the basis that the reaction stoichiometry between AA and O(2) is 1:1 when the Abeta concentration is kept at a much greater value than that of Cu(II). A mechanism is proposed for the AA oxidation in which the oxidation states of the copper center in the Abeta complex alternates between 2+ and 1+. The catalytic activity of Cu(II) toward O(2) reduction was found to decrease in the order of free Cu(II) > Abeta(1-16)-Cu(II) > Abeta(1-42)-Cu(II) > Cu(II) complexed by the Abeta oligomer/fibril mixture > Cu(II) in Abeta fibrils. The finding that Cu(II) in oligomeric and fibrous Abeta aggregates possesses considerable activity toward H(2)O(2) generation is particularly significant, since in senile plaques of AD patients the coexisting copper and Abeta aggregates have been suggested to inflict oxidative stress through the production of reactive oxygen species (ROS). Although Cu(II) bound to oligomeric and fibrous Abeta aggregates is less effective than free Cu(II) and the monomeric Abeta-Cu(II) complex in producing ROS, in vivo the Cu(II)-containing Abeta oligomers and fibrils might be more biologically relevant given their stronger association with cell membranes and the closer proximity of ROS to cell membranes.
Mass spectrometry imaging (MSI) is an emerging technology for high-resolution plant biology. It has been utilized to study plant-pest interactions, but limited to the surface interfaces. Here we ...expand the technology to explore the chemical interactions occurring inside the plant tissues. Two sample preparation methods, imprinting and fracturing, were developed and applied, for the first time, to visualize internal metabolites of leaves in matrix-assisted laser desorption ionization (MALDI)-MSI. This is also the first time nanoparticle-based ionization was implemented to ionize diterpenoid phytochemicals that were difficult to analyze with traditional organic matrices. The interactions between rice-bacterium and soybean-aphid were investigated as two model systems to demonstrate the capability of high-resolution MSI based on MALDI. Localized molecular information on various plant- or pest-derived chemicals provided valuable insight for the molecular processes occurring during the plant-pest interactions. Specifically, salicylic acid and isoflavone based resistance was visualized in the soybean-aphid system and antibiotic diterpenoids in rice-bacterium interactions.
Plant tissues present intriguing systems for study by mass spectrometry imaging, as they exhibit a complex metabolism and a high degree of spatial localization. This chapter presents a methodology ...for preparation of plant tissue sections for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) analysis and the use of a hybrid mass spectrometer for "multiplex" imaging. The multiplex method described here provides a wide range of analytical information, including high-resolution, accurate mass imaging and tandem MS scans for structural information, all within a single experiment. While this procedure was developed for plant tissues, it can be readily adapted for analysis of other sample types.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a widely adopted, versatile technique, especially in high-throughput analysis and imaging. However, matrix-dependent ...selectivity of analytes is often a severe limitation. In this work, a mixture of organic 2,5-dihydroxybenzoic acid and inorganic Fe
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nanoparticles is developed as a binary MALDI matrix to alleviate the well-known issue of triacylglycerol (TG) ion suppression by phosphatidylcholine (PC). In application to lipid standards and maize seed cross-sections, the binary matrix not only dramatically reduced the ion suppression of TG, but also efficiently desorbed and ionized a wide variety of lipids such as cationic PC, anionic phosphatidylethanolamine (PE) and phosphatidylinositol (PI), and neutral digalactosyldiacylglycerol (DGDG). The binary matrix was also very efficient for large polysaccharides, which were not detected by either of the individual matrices. The usefulness of the binary matrix is demonstrated in MS imaging of maize seed sections, successfully visualizing diverse medium-size molecules and acquiring high-quality MS/MS spectra for these compounds.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a widely adopted, versatile technique, especially in high-throughput analysis and imaging. However, matrix-dependent ...selectivity of analytes is often a severe limitation. In this work, a mixture of organic 2,5-dihydroxybenzoic acid and inorganic Fe
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nanoparticles is developed as a binary MALDI matrix to alleviate the well-known issue of triacylglycerol (TG) ion suppression by phosphatidylcholine (PC). In application to lipid standards and maize seed cross-sections, the binary matrix not only dramatically reduced the ion suppression of TG, but also efficiently desorbed and ionized a wide variety of lipids such as cationic PC, anionic phosphatidylethanolamine (PE) and phosphatidylinositol (PI), and neutral digalactosyldiacylglycerol (DGDG). The binary matrix was also very efficient for large polysaccharides, which were not detected by either of the individual matrices. The usefulness of the binary matrix is demonstrated in MS imaging of maize seed sections, successfully visualizing diverse medium-size molecules and acquiring high-quality MS/MS spectra for these compounds.
A binary matrix of DHB and Fe
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NPs provides broad coverage for medium size metabolites.
The main goal of the presented research is development of nanoparticle based matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). This dissertation includes the application of ...previously developed data acquisition methods, development of novel sample preparation methods, application and comparison of novel nanoparticle matrices, and comparison of two nanoparticle matrix application methods for MALDI-MS and MALDI-MS imaging. Chapter 1 presents background information about MALDI-MS and a general workflow for MALDI-MS imaging experiments. Chapter 6 provides a summary of the presented work and possible future directions. Chapter 2 describes a “multiplex” MALDI-MS imaging technique applied to study latent human fingerprints. In this study, we applied previously developed multiplex data acquisition techniques for the detection of endogenous and exogenous compounds with structural information in a single MALDI-MS imaging experiment. This study also depicts the importance of the sublimation matrix application method for analysis of very delicate samples such as latent fingerprints. Chapter 3 presents the development of novel sample preparation methods for nanoparticle-assisted laser desorption/ionization-mass spectrometry -mass spectrometry (NALDI-MS) imaging of metabolites in plant tissues. In this chapter, we developed a novel fracturing method for MALDI-MS/ NALDI-MS imaging of rice leaf to study a plant-pathogen interactions. We successfully detected and imaged diterpenoids produced against the Xanthomonas oryzae pv. oryzae infection in rice leaves. Chapter 4 is focused on novel nanoparticle (NP) matrix development for NALDI-MS of low-molecular weight plant metabolites. This large-scale NP screening was inspired by the success of the metabolite detection/imaging in Chapter 3. In this work, we studied three different classes of NPs for desorption/ionization of a wide variety of plant metabolites and sought to shed some light on the NALDI mechanism. Chapter 5 is a further extension of the study in Chapter 4, in which we compare two NP application methods for efficiency of desorption/ionization of plant metabolites and homogeneity of application. We compare application of a nanoparticle suspension by oscillating capillary nebulizer and solvent-free sputter coating of metal NPs. The work presented in Chapters 4 and 5 will be helpful for development of novel matrices for analysis and imaging of low-molecular weight metabolites that are difficult to ionize using traditional MALDI-MS matrices.
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