Seeing the forest for the trees : retrieving plant secondary biochemical pathways from metabolome networks

Over the last decade, a giant leap forward has been made in resolving the main bottleneck in metabolomics, i.e., the structural characterization of the many unknowns. This has led to the next challenge in this research field: retrieving biochemical pathway information from the various types of networks that can be constructed from metabolome data. Searching putative biochemical pathways, referred to as biotransformation paths, is complicated because several flaws occur during the construction of metabolome networks. Multiple network analysis tools have been developed to deal with these flaws, while in silico retrosynthesis is appearing as an alternative approach. In this review, the different types of metabolome networks, their flaws, and the various tools to trace these biotransformation paths are discussed

Novel methods for the analysis of small molecule fragmentation mass spectra

The identification of small molecules, such as metabolites, in a high throughput manner plays an important in many research areas. Mass spectrometry (MS) is one of the predominant analysis technologies and is much more sensitive than nuclear magnetic resonance spectroscopy. Fragmentation of the molecules is used to obtain information beyond its mass. Gas chromatography-MS is one of the oldest and most widespread techniques for the analysis of small molecules. Commonly, the molecule is fragmented using electron ionization (EI). Using this technique, the molecular ion peak is often barely visible in the mass spectrum or even absent. We present a method to calculate fragmentation trees from high mass accuracy EI spectra, which annotate the peaks in the mass spectrum with molecular formulas of fragments and explain relevant fragmentation pathways. Fragmentation trees enable the identification of the molecular ion and its molecular formula if the molecular ion is present in the spectrum. The method works even if the molecular ion is of very low abundance. MS experts confirm that the calculated trees correspond very well to known fragmentation mechanisms.Using pairwise local alignments of fragmentation trees, structural and chemical similarities to already-known molecules can be determined. In order to compare a fragmentation tree of an unknown metabolite to a huge database of fragmentation trees, fast algorithms for solving the tree alignment problem are required. Unfortunately the alignment of unordered trees, such as fragmentation trees, is NP-hard. We present three exact algorithms for the problem. Evaluation of our methods showed that thousands of alignments can be computed in a matter of minutes. Both the computation and the comparison of fragmentation trees are rule-free approaches that require no chemical knowledge about the unknown molecule and thus will be very helpful in the automated analysis of metabolites that are not included in common libraries

Examining lipid metabolism of colorectal adenomas and carcinomas using Rapid Evaporative Ionisation Mass Spectrometry (REIMS)

Background There is an unmet need for real-time intraoperative colorectal tissue recognition, which would promote personalised oncologic decision making. Rapid Evaporative Ionization Mass Spectrometry (REIMS) analyses the composition of cellular lipids through the aerosol generated from electrosurgical instruments, providing a novel diagnostic platform and surgeon feedback. Thesis Hypothesis Colorectal lipid metabolism and cellular lipid composition are associated with the phenotype of colorectal adenomas and carcinomas, which can be leveraged for tissue recognition in vivo. Methods This thesis contains three work packages. First, a method for REIMS spectral quality control was developed based on a human dataset and analysis of a porcine model assessed the spectral impact of technical and environmental factors. Second, an ex vivo spectral reference database was constructed from analysis of human colorectal tissues, assessing the ability of REIMS for tissue recognition. Finally, REIMS was translated into the operating theatre, for proof-of-principle application of during transanal minimally invasive surgery (TAMIS). Results Sensitivity analyses revealed seven minimum quality criteria for REIMS spectra to be included in all future statistical analyses, with quality also impacted by low diathermy power, coagulation mode and tissue contamination. Based on tissue of 161 patients, REIMS could differentiate colorectal normal, adenoma and cancer tissue with 91.1% accuracy, and disease from normal with 93.5% accuracy. REIMS could risk-stratify adenomas by predicting grade of dysplasia, however not histological features of poor prognosis in cancers. 61 pertinent lipid metabolites were structurally identified. REIMS was coupled to TAMIS in seven patients. Optimisation of the workflow successfully increased signal intensity, with tissue recognition showing high accuracy in vivo and identification of a cancer-involved margin. Discussion This thesis demonstrates that REIMS can be optimised and applied for accurate real-time colorectal tissue recognition based on cellular lipid composition. This can be translated in vivo, with promising results during first-in-man mass spectrometry-coupled TAMIS.Open Acces

Mass Spectrometric Studies on the Primary Products of Fast Pyrolysis of Carbohydrates and the Molecular Structures of Asphaltenes, and the Development of a Rastering Probe for Laser-Induced Acoustic Desorption into an Atmospheric Pressure Chemical Ionization Source

Mass spectrometry (MS) has proven invaluable in the field of mixture analysis and structural elucidation. Tandem mass spectrometry (MS/MS) utilizing collision-activated dissociation (CAD) has become the technique of choice for structural elucidation of unknown analytes in mixtures. When coupled with gas chromatography (GC) or high performance liquid chromatography (HPLC), it allows for trace level analysis of mixture components. In spite of the utility of mass spectrometry in complex mixture analysis, it does have limitations. Traditional GC/MS methods used for the analysis of fast pyrolysis products cannot be used to analyze the primary products, thus limiting the knowledge that can be obtained regarding the true mechanisms of fast pyrolysis, ultimately restricting the level of control over what final products are formed. Analysis of mixtures of hydrocarbons, such as crude oil, is also still a problematic area for mass spectrometry due to the lack of suitable evaporation/ionization methods for the heavier components. Consequently, very little is known about the structures of molecules in asphaltenes, the heaviest fraction of crude oil and one of the most complex mixtures in nature. Elucidating the structures of the compounds present in these mixtures is important for the rational design of methods to prevent the problems the cause. Experiments described in this thesis employed tandem mass spectrometry to achieve a better understanding of the primary products of fast pyrolysis of carbohydrates and structures of molecules in asphaltenes. Chapter 2 briefly describes the instrumentation used for the research presented in this dissertation. Chapter 3 discusses the development of on-line mass spectrometric methods for the determination of the primary products of fast pyrolysis of carbohydrates and their gas- phase reactivity, demonstrating that there are many primary products that cannot be analyzed using traditional methods. Chapter 4 examines the differences in the molecular structures of petroleum and coal asphaltenes. Chapter 5 focuses on changes to asphaltenes\u27 molecular structures when they are subjected to the hydrocracking process, a common practice in crude oil refinement. Chapter 6 compares field deposit asphaltenes, removed from a pipeline, to heptane precipitated asphaltenes from crude oil in a laboratory. Chapter 7 contrasts the effects of using different solvents in atmospheric pressure chemical ionization (APCI) of asphaltenes. Chapters 8 and 9 focus on advances for laser-induced acoustic desorption (LIAD). Chapter 8 discusses improvements LIAD/APCI, including the development of a high-power laser probe for more reproducible evaporation of high-mass compounds into the gas phase, and the development of a rastering assembly that greatly increases the surface area of the LIAD foil that can be sampled. Chapter 9 discusses a novel chamber for preparing sample foils for LIAD by using a drying gas to prepare foils. The new chamber that helps in the production of foils with a more uniform sample layer than previously possible for nonpolar analytes to improve the reproducibility of LIAD

Advances in Nuclear Magnetic Resonance for Drug Discovery

Background—Drug discovery is a complex and unpredictable endeavor with a high failure rate. Current trends in the pharmaceutical industry have exasperated these challenges and are contributing to the dramatic decline in productivity observed over the last decade. The industrialization of science by forcing the drug discovery process to adhere to assembly-line protocols is imposing unnecessary restrictions, such as short project time-lines. Recent advances in nuclear magnetic resonance are responding to these self-imposed limitations and are providing opportunities to increase the success rate of drug discovery. Objective/Method—A review of recent advancements in NMR technology that have the potential of significantly impacting and benefiting the drug discovery process will be presented. These include fast NMR data collection protocols and high-throughput protein structure determination, rapid protein-ligand co-structure determination, lead discovery using fragment-based NMR affinity screens, NMR metabolomics to monitor in vivo efficacy and toxicity for lead compounds, and the identification of new therapeutic targets through the functional annotation of proteins by FASTNMR. Conclusion—NMR is a critical component of the drug discovery process, where the versatility of the technique enables it to continually expand and evolve its role. NMR is expected to maintain this growth over the next decade with advancements in automation, speed of structure calculation, incell imaging techniques, and the expansion of NMR amenable targets

ECOMICS: A Web-Based Toolkit for Investigating the Biomolecular Web in Ecosystems Using a Trans-omics Approach

Ecosystems can be conceptually thought of as interconnected environmental and metabolic systems, in which small molecules to macro-molecules interact through diverse networks. State-of-the-art technologies in post-genomic science offer ways to inspect and analyze this biomolecular web using omics-based approaches. Exploring useful genes and enzymes, as well as biomass resources responsible for anabolism and catabolism within ecosystems will contribute to a better understanding of environmental functions and their application to biotechnology. Here we present ECOMICS, a suite of web-based tools for ECosystem trans-OMICS investigation that target metagenomic, metatranscriptomic, and meta-metabolomic systems, including biomacromolecular mixtures derived from biomass. ECOMICS is made of four integrated webtools. E-class allows for the sequence-based taxonomic classification of eukaryotic and prokaryotic ribosomal data and the functional classification of selected enzymes. FT2B allows for the digital processing of NMR spectra for downstream metabolic or chemical phenotyping. Bm-Char allows for statistical assignment of specific compounds found in lignocellulose-based biomass, and HetMap is a data matrix generator and correlation calculator that can be applied to trans-omics datasets as analyzed by these and other web tools. This web suite is unique in that it allows for the monitoring of biomass metabolism in a particular environment, i.e., from macromolecular complexes (FT2DB and Bm-Char) to microbial composition and degradation (E-class), and makes possible the understanding of relationships between molecular and microbial elements (HetMap). This website is available to the public domain at: https://database.riken.jp/ecomics/

Next Generation Optical Analysis for Agrochemical Research & Development

The world’s population is increasing rapidly and higher calorific diets are becoming more common; as a consequence the demand for grain is predicted to increase by more than 50% by 2050 without a significant increase in the available agricultural land. Maximising the productivity of the existing agricultural land is key to maintaining food security and agrochemicals continue to be a key enabler for the efficiency gains required. However, agrochemicals can be susceptible to significant losses and thus often require further chemical to be applied to compensate. Sources of such losses include spray drift, poor spray retention/capture by the target and poor penetration through the plant cuticle. The effectiveness of a crop protection agent depends not only on the intrinsic activity of the active ingredient (AI) but also on the physicochemical properties of the formulation. These properties can be modified by using formulation components, known as adjuvants, which can be used to help mitigate such losses. Adjuvants exert their effects by, for example, controlling droplet size and distribution through their effect on surface tension which can also improve penetration into leaves through the cuticle wax which coats the epidermis of leaves and acts as a protective barrier. However, characterising how they alter the movement of the AIs can be challenging. Optical techniques have shown promise in a multitude of scientifically related areas, such as in vivo tissue imaging, but none have yet been applied to aiding the agrochemical industry. By probing the interactions between leaf surface and agrochemical agent, with light, one is able to obtain a large amount of diagnostic information, non-invasively. Whereas techniques like Raman 3 spectroscopy are limited by long acquisition times, coherent Raman techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) are coherently driven and provide an enhanced signal, and also allow for video-rate imaging. In this thesis, I have applied this cutting-edge laser imaging technique as a novel analytical technique that allows the in situ analysis of agrochemicals in living plant tissues at a cellular level. In Chapters 4 through 7, multiple factors essential for a functional and efficient agrochemical were considered and experimented. In Chapter 4, a typical industry study highlights the need for innovative and rapid technologies in the agrochemical industry. The resulting chapters (5, 6, and 7) outline several ways in which Coherent Raman Scattering (CRS) techniques can improve the current capabilities of agrochemical testing. By utilising a model system, paraffin wax, a cheap and rapid protocol can provide accurate diffusion information and repeatable results. Chapters 6 and 7 use both this protocol to gain comparative data on several adjuvants and active ingredients in paraffin wax and in vivo, in a variety of plants. The ability to visualise agrochemical products on a leaf surface to reveal interactions between the materials of the product and with the leaf surface will enable a step change in the agrochemical design process, through determination of the spatial distribution of the materials and their roles within the applied products. It is hoped that the technology developed in this thesis could play a big role in the development of future agrochemical products that are tailored to maximise efficacy and minimise environmental impact

Ion Mobility-Mass Spectrometry and Collision Induced Unfolding of Multi-Protein Ligand Complexes.

Mass spectrometry (MS) serves as an indispensable technology for modern pharmaceutical drug discovery and development processes, where it is used to assess ligand binding to target proteins and to search for biomarkers that can be used to gauge disease progression and drug action. However, MS is rarely treated as a screening technology for the structural consequences of drug binding. Instead, more time-consuming technologies capable of projecting atomic models of protein-drug interactions are utilized. In this thesis, ion mobility-mass spectrometry (IM-MS) methods are developed in order to fill these technology gaps. Principle among these is collision induced unfolding (CIU), which leverages the ability of IM to separate ions according to their size and charge, in order to fingerprint gas-phase unfolding pathways for non-covalent protein complexes. Following a comprehensive introductory chapter, we demonstrate the consequences of sugar binding on the CIU of Concanavalin A (Con A) in Chapter 2. Our CIU assay reveals cooperative stabilization upon small molecule binding, and such effect cannot be easily detected by solution phase assays, or by MS alone. In Chapter 3, the underlying mechanism of multi-protein unfolding is systematically investigated by IM-MS and molecular modeling approaches. Our results show a strong positive correlation between monomeric Coulombic unfolding and the tetrameric CIU process. This provides strong evidence that multi-protein unfolding events are initiated primarily by charge migration from the complex to a single monomer. In Chapter 4, the interactions between human histone deacetylase 8 (HDAC8) and poly-r(C)-binding protein 1 (PCBP1) are investigated by IM-MS. Our data suggest that these proteins interact with each other in a specific manner, a fact revealed by our optimized ESI-MS workflow for quantifying binding affinity (KD) for weakly-associated hetero-protein complexes. In Chapter 5, the translocator protein (TSPO) dimer from Rhodobacter sphaeroides, as well as its disease-associated variant forms, is analyzed by IM-MS and CIU assays. By utilizing a combination of CIU and collision induced dissociation (CID) stability data, an unknown endogenous ligand bound to TSPO is detected and identified.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116693/1/shuainiu_1.pd