Fluorescent labeling is a method used for visualization of various types of biomolecules including proteins and protein complexes. However, the effect of protein labeling on protein structure and functions has not been investigated so far. The goal of the diploma thesis was to examine an influence of NHS-fluorescein binding on structure and function of human carbonic anhydrase I (hCA-I). The particular aims of this work were to prepare recombinant 15N-hCA-I which was used for NMR structure analysis of carbonic anhydrase upon fluorescent labeling. Furthermore, enzyme activity was measured in order to find out a correlation between the concentration of NHS- fluorescein and protein function. In addition, the reaction mixtures were systematically analyzed by ESI FT-ICR mass spectrometry. The analysis revealed experimental conditions for fluorescent labeling of human carbonic anhydrase I with minimal effect on protein structure and function. The results of this study show that the calculation of molar excess of NHS-fluorescein cannot rely on a simple procedure provided by manufacturer. However, due to decrease of enzyme activity upon fluorescent labeling, it is better to take into count the influence of NHS-fluorescein concentration on the relative enzymatic activity. Moreover, the calculation of molar...

Dissolved organic matter (DOM) is a central component of the global carbon cycle. Thus, small changes to the amount of DOM imported, processed and produced within lakes can have a large effect on regional carbon budgets. In addition to being a vital energy source at the base of the aquatic food web, DOM is physico-chemically reactive. However, identifying and understanding the controls of DOM processing has remained challenging due to the complex composition of DOM. DOM comprises a mixture of decomposition by-products of terrestrial origin as well as newly synthesized material from in situ production. DOM compounds form gradients of reactivity to biogeochemical processes, such as photodegradation, biodegradation, and flocculation, and they perform a suite of functions in aquatic systems. The overarching goal of this thesis was to investigate controls of DOM processing in Swedish lakes. We do this in two ways: 1) by characterizing the molecular-level composition of DOM in lakes, and 2) by investigating interactions between very labile and relatively recalcitrant DOM. The first three chapters utilize ultrahigh resolution mass spectrometry to show that the detailed chemical composition of DOM varies along a hydrology gradient, and secondarily along a temperature gradient that co-varies with agriculture and nutrients. Next, we illustrate the coherence between molecular-level characteristics and bulk optical characteristics. Together, these studies suggest that protein-like fluorescence, aliphatic compounds, and N-containing compounds are either resistant to degradation or tightly cycled in the system, and thus persist at long water residence times. The most oxidized compounds, such as vascular plant-derived polyphenolic compounds, are abundant in areas with high precipitation and are lost with increasing water residence time. Vascular plant-derived polyphenolic compounds were most strongly related to DOM with high apparent molecular weight, suggesting that hydrophobic interactions drive aggregate formation. Furthermore, the association of high molecular weight DOM with polyphenolic compounds suggests that aggregates are hotspots of reactivity in aquatic systems. Finally, we find no indication that the addition of labile organic matter enhances the biodegradation of less reactive DOM. Thus, we suggest that in freshwaters, intrinsic molecular properties, such as the basic structural features of compounds, dominate over extrinsic factors.

The interplay of rhizosphere components such as root exudates, microbes, and minerals results in small-scale gradients of organic molecules in the soil around roots. The current methods for the direct chemical imaging of plant metabolites in the rhizosphere often lack molecular information or require labeling with fluorescent tags or isotopes. Here, we present a novel workflow using laser desorption ionization (LDI) combined with mass spectrometric imaging (MSI) to directly analyze plant metabolites in a complex soil matrix. Undisturbed samples of the roots and the surrounding soil of Zea mays L. plants from either field- or laboratory-scale experiments were embedded and cryosectioned to 100 mm thin sections. The target metabolites were detected with a spatial resolution of 25 mm in the root and the surrounding soil based on accurate masses using ultra-high mass resolution laser desorption ionization Fourier-transform ion cyclotron resonance mass spectrometry (LDI-FT-ICR-MS). Using this workflow, we could determine the rhizosphere gradients of a dihexose (e.g., sucrose) and other plant metabolites (e.g., coumaric acid, vanillic acid). The molecular gradients for the dihexose showed a high abundance of this metabolite in the root and a strong depletion of the signal intensity within 150 mm from the root surface. Analyzing several sections from the same undisturbed soil sample allowed us to follow molecular gradients along the root axis. Benefiting from the ultra-high mass resolution, isotopologues of the dihexose could be readily resolved to enable the detection of stable isotope labels on the compound level. Overall, the direct molecular imaging via LDI-FT-ICR-MS allows for the first time a nontargeted or targeted analysis of plant metabolites in undisturbed soil samples, paving the way to study the turnover of root-derived organic carbon in the rhizosphere with high chemical and spatial resolution.

No description available.

Dissolved organic matter (DOM) is the largest pool of organic carbon in aquatic systems and an important component of the global carbon cycle. Large amounts of DOM are decomposed within lakes, resulting in fluxes of CO2 and CH4 to the atmosphere. Therefore, there is a considerable interest in understanding the controls of DOM decomposition in freshwaters. There is evidence that in lakes intrinsic controls related to DOM composition are of primary importance, yet our knowledge about molecular drivers of DOM degradation is limited. This thesis addresses the link between chemical composition and reactivity of lake DOM by applying an experimental approach, molecular-level DOM characterization, and kinetic modeling of DOM decay. The first study shows that photoinduced transformations and partial removal of colored aromatic components of DOM have profound effects on DOM degradation kinetics, mediated by the shifts in the relative share of rapidly and slowly degrading DOM fractions. Two following studies estimate exponential decay coefficients for each individual molecular formula identified within bulk DOM. A continuous distribution of exponential decay coefficients is found within bulk DOM, which directly corroborates the central and previously empirically untested assumption behind the reactivity continuum model of DOM decay. Further, individual decay rates are evaluated in connection to specific molecular properties. On average, highly unsaturated and phenolic compounds appear to be more persistent than compounds with higher aromatic content (plant polyphenols and polycondensed aromatics), and aliphatic compounds demonstrate the highest decay rates. The reactivity of aromatics additionally increases with increasing nominal oxidation state of carbon. Molecular analysis further indicates that increasing reactivity of DOM after UV exposure is caused by disintegration of supramolecular complexes. Study IV shows that changes in relative proportion of terrestrial versus algal DOM control degradability of DOM through seasons. Under ice, when algal-derived DOM is maximally depleted, DOM degradation potential converges to similarly low levels, regardless of lake type (productive or humic), and bacterial respiration primarily relies on terrestrial carbon. This suggests a general pattern of baseline metabolism across boreal lakes. I conclude that DOM is a dynamic reactivity continuum and a tight link exists between DOM behavior and compositional properties.

Analytical Characterization of complex mixtures, such as crude oil, environmental samples, and biological mixtures, is challenging because of the large diversity of molecular components. Mass spectrometry based techniques are among the most powerful tools for the separation of molecules based on their molecular composition, and the coupling of ion mobility spectrometry has enabled the separation and structural elucidation using the tridimensional structure of the molecule. The present work expands the ability of analytical chemists by furthering the development of IMS-MS instrumentation by coupling Trapped Ion Mobility Spectrometry to Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (TIMS-FT-ICR MS). The TIMS-FT-ICR MS platform combines the high-resolution separation of TIMS, which has mobility resolving powers up to 400, and ultra-high mass resolution of FT-ICR MS, with mass resolving power over 1,000,000. This instrumentation allows the assignment of exact chemical composition for compounds in a complex mixture, as well as measurement of the collision cross-section of the molecule. Herein, the principles of the TIMS separation and its coupling to FT-ICR MS are described, as well as how the platform can be applied to targeted analysis of molecules, and untargeted characterization of complex mixtures. Molecular standards were analyzed by TIMS-MS in order to develop a computational workflow that can be utilized to elucidate molecular structure, using the measured collision cross-section of the ion. This workflow enabled identification of structural, cis/trans isomers, and chelated molecules and provides the basis for unsupervised structural elucidation of a complex mixture, and in particular for the elucidation of hydrocarbons from fossil fuels. In summary, this work presents the coupling of TIMS-FT-ICR MS and provides examples of applications as a proof of concept of the potential of this platform for solving complex analytical challenges.

Mass-selected infrared multiple photon spectroscopy (IRMPD), Fourier transform ion cyclotron resonance (FT-ICR) kinetic experiments, RRKM and electronic structure calculations have been performed in order to propose a complex mechanism involving the formation of the proton-bound dimer of water (H5O2+) from 1,1,3,3-tetrafluorodimethyl ether. It has been found that the reaction is facilitated by a series of sequential exothermic bimolecular ion-molecule reactions. Evidence for the dominant mechanistic pathway involving the reaction of CF2H-O=CHF+, an ion of m/z 99, with water is presented. The primary channel occurs via nucleophilic attack of water on the ion of m/z 99 (CF2H-O=CHF+), to lose formyl fluoride and yield protonated difluoromethanol (m/z 69). Association of a second water molecule with protonated difluoromethanol generates a reactive intermediate which decomposes via a 1,4-elimination to release hydrogen fluoride and yield the proton-bound dimer of water and formyl fluoride (m/z 67). The 1,4-elimination of hydrogen fluoride is found to be strongly supported by the results of both RRKM theory and electronic structure calculations. Lastly, the elimination of formyl fluoride occurs by the association of a third water molecule to produce H5O2+ (m/z 37). The most probable isomeric forms of the ions with m/z 99 and 69 were found using IRMPD spectroscopy and electronic structure theory calculations. Thermochemical information for reactant, transition and product species was obtained using MP2/aug-cc-pVQZ//MP2(full)/6-31G(d) level of theory. Ionic hydrogen bond (IHB) interactions, resulting from the association of ammonia and two of the protonated methylxanthine derivatives, caffeine and theophylline, have been characterized using mass-selected IRMPD and electronic structure calculations at the MP2/aug-cc-pVTZ//B3LYP/6-311+G(d,p) level of theory. It was found that the formation of a proton-bound dimer (PBD) of caffeine and ammonia was elusive under the experimental conditions. The low binding energy of the caffeine and ammonia PBD is responsible for the perceived difficulty in obtaining an IRMPD spectrum. The IRMPD spectrum of the PBD of theophylline and ammonia was obtained and revealed bidentate IHB formation within the complex, which greatly increased the binding energy relative to the most stable isomer of the PBD of caffeine and ammonia. The IRMPD spectra of the protonated forms of caffeine and theophylline have also obtained. The spectrum of protonated caffeine showed the dominant existence of a single isomer, whereas the spectrum of protonated theophylline showed a mixture of isomers. The mixture of isomers of protonated theophylline resulted as a consequence of proton-transport catalysis (PTC) occurring within the PBD of theophylline and ammonia. All calculated harmonic spectra have been produced at the B3LYP/6-311+G(d,p) level of theory with fundamental frequencies scaled by 0.9679; calculated anharmonic spectra have also been provided at the same level of theory and were found to greatly improve the match with the IRMPD spectra obtained in all cases. Ionic hydrogen bond (IHB) interactions, resulting from the association of caffeine and theophylline with their protonated counterparts, forming proton-bound homodimers, have been characterized using mass-selected IRMPD and electronic structure calculations at the MP2/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) level of theory. It is found that the IRMPD spectra of the proton-bound homodimers of caffeine and theophylline are complicated resulting from the existence of several pairs of enantiomers separated by a narrow range of relative Gibbs free energies (298 K) of 15.6 and 18.2 kJ mol-1, respectively. The IRMPD spectrum of the proton-bound homodimer of theophylline is dominated by a unique isomer facilitated by formation of a bidentate IHB. Formation of this interaction lowers the relative Gibbs free energy of the ion to 9.75 kJ mol-1 below that of the most favourable pair of enantiomers. The IRMPD spectrum of the PBD of caffeine is complicated by the existence of at least two pairs of enantiomers with the strong likelihood of the spectral contributions of a third pair existing. The most favourable enantiomeric pair involves the formation of a O-H+⋯O IHB. However, verification of a pair of enantiomeric PBDs containing a N-H+⋯O IHB is also observed in the IRMPD spectrum of the PBD of caffeine due to the presence of three free carbonyl stretching modes located at 1731, 1751 and 1785 cm-1. The mass-selected IRMPD spectra of the sodium cation-bound dimers (SCBD) of caffeine and theophylline also have been obtained. Both the mass-selected IRMPD spectra and electronic structure calculations predict the most likely structure of the SCBDs of caffeine and theophylline to form by an efficient O⋯Na+⋯O interaction between C=O functional groups possessed by each monomer. The frequencies of the C=O-Na+ stretch are found to be nearly identical in the IRMPD spectra for both of the SCBDs of caffeine and theophylline at 1644 and 1646 cm-1, respectively. However, the degenerate free C=O symmetric and asymmetric stretches for the SCBDs of caffeine and theophylline found at 1732 and 1758 cm^(-1), respectively, demonstrating a red-shift for caffeine possibly linked to a steric interaction absent in theophylline. Free rotation about the O⋯Na+⋯O bond is found to greatly decrease the complexity of the IRMPD spectra of the SCBDs of caffeine and theophylline and demonstrates excellent agreement between the IRMPD and calculated spectra. Electronic structure calculations have been done at the MP2(full)/aug-cc-pCVTZ/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) level of theory using the aug-cc-pCVTZ basis set for Na+ and all Na+-interacting heterotatoms, and the 6-311+G(2d,2p) basis set for all non-interacting atoms within the SCBDs, in order to provide accurate electronic energies. Currently, installation and implementation of a pulsed electrospray high pressure ion source mated to an existing high pressure mass spectrometer (HPMS) is underway. The new ion source will greatly increase the range of possibilities for the study of ion-molecule reactions in the McMahon laboratory. One of the unique features of the new design is the incorporation of a gas-tight electrospray interface, allowing for more possibilities than only the study of cluster-ion equilibria involving hydration (H2On⋯S+), where S+ is an ion produced by electrospray. Other small prototypical biological molecules such as amines and thiols can be used without concern for the toxicity of these species. Another unique design feature allows electrosprayed ions to associate with neutral solvent species in an electric field free reaction chamber (RC). This ensures that values of equilibrium constants determined are truly representative of ions in states of thermochemical equilibrium. The existing HPMS in the McMahon laboratory is limited to the study of small volatile organic molecules. The new ion source will permit the exploration of systems involving non-volatile species, doubly charged ions and many biologically relevant molecules such as amino acids, peptides, nucleobases and carbohydrates.

Mass-selected infrared multiple photon spectroscopy (IRMPD), Fourier transform ion cyclotron resonance (FT-ICR) kinetic experiments, RRKM and electronic structure calculations have been performed in order to propose a complex mechanism involving the formation of the proton-bound dimer of water (H5O2+) from 1,1,3,3-tetrafluorodimethyl ether. It has been found that the reaction is facilitated by a series of sequential exothermic bimolecular ion-molecule reactions. Evidence for the dominant mechanistic pathway involving the reaction of CF2H-O=CHF+, an ion of m/z 99, with water is presented. The primary channel occurs via nucleophilic attack of water on the ion of m/z 99 (CF2H-O=CHF+), to lose formyl fluoride and yield protonated difluoromethanol (m/z 69). Association of a second water molecule with protonated difluoromethanol generates a reactive intermediate which decomposes via a 1,4-elimination to release hydrogen fluoride and yield the proton-bound dimer of water and formyl fluoride (m/z 67). The 1,4-elimination of hydrogen fluoride is found to be strongly supported by the results of both RRKM theory and electronic structure calculations. Lastly, the elimination of formyl fluoride occurs by the association of a third water molecule to produce H5O2+ (m/z 37). The most probable isomeric forms of the ions with m/z 99 and 69 were found using IRMPD spectroscopy and electronic structure theory calculations. Thermochemical information for reactant, transition and product species was obtained using MP2/aug-cc-pVQZ//MP2(full)/6-31G(d) level of theory. Ionic hydrogen bond (IHB) interactions, resulting from the association of ammonia and two of the protonated methylxanthine derivatives, caffeine and theophylline, have been characterized using mass-selected IRMPD and electronic structure calculations at the MP2/aug-cc-pVTZ//B3LYP/6-311+G(d,p) level of theory. It was found that the formation of a proton-bound dimer (PBD) of caffeine and ammonia was elusive under the experimental conditions. The low binding energy of the caffeine and ammonia PBD is responsible for the perceived difficulty in obtaining an IRMPD spectrum. The IRMPD spectrum of the PBD of theophylline and ammonia was obtained and revealed bidentate IHB formation within the complex, which greatly increased the binding energy relative to the most stable isomer of the PBD of caffeine and ammonia. The IRMPD spectra of the protonated forms of caffeine and theophylline have also obtained. The spectrum of protonated caffeine showed the dominant existence of a single isomer, whereas the spectrum of protonated theophylline showed a mixture of isomers. The mixture of isomers of protonated theophylline resulted as a consequence of proton-transport catalysis (PTC) occurring within the PBD of theophylline and ammonia. All calculated harmonic spectra have been produced at the B3LYP/6-311+G(d,p) level of theory with fundamental frequencies scaled by 0.9679; calculated anharmonic spectra have also been provided at the same level of theory and were found to greatly improve the match with the IRMPD spectra obtained in all cases. Ionic hydrogen bond (IHB) interactions, resulting from the association of caffeine and theophylline with their protonated counterparts, forming proton-bound homodimers, have been characterized using mass-selected IRMPD and electronic structure calculations at the MP2/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) level of theory. It is found that the IRMPD spectra of the proton-bound homodimers of caffeine and theophylline are complicated resulting from the existence of several pairs of enantiomers separated by a narrow range of relative Gibbs free energies (298 K) of 15.6 and 18.2 kJ mol-1, respectively. The IRMPD spectrum of the proton-bound homodimer of theophylline is dominated by a unique isomer facilitated by formation of a bidentate IHB. Formation of this interaction lowers the relative Gibbs free energy of the ion to 9.75 kJ mol-1 below that of the most favourable pair of enantiomers. The IRMPD spectrum of the PBD of caffeine is complicated by the existence of at least two pairs of enantiomers with the strong likelihood of the spectral contributions of a third pair existing. The most favourable enantiomeric pair involves the formation of a O-H+⋯O IHB. However, verification of a pair of enantiomeric PBDs containing a N-H+⋯O IHB is also observed in the IRMPD spectrum of the PBD of caffeine due to the presence of three free carbonyl stretching modes located at 1731, 1751 and 1785 cm-1. The mass-selected IRMPD spectra of the sodium cation-bound dimers (SCBD) of caffeine and theophylline also have been obtained. Both the mass-selected IRMPD spectra and electronic structure calculations predict the most likely structure of the SCBDs of caffeine and theophylline to form by an efficient O⋯Na+⋯O interaction between C=O functional groups possessed by each monomer. The frequencies of the C=O-Na+ stretch are found to be nearly identical in the IRMPD spectra for both of the SCBDs of caffeine and theophylline at 1644 and 1646 cm-1, respectively. However, the degenerate free C=O symmetric and asymmetric stretches for the SCBDs of caffeine and theophylline found at 1732 and 1758 cm^(-1), respectively, demonstrating a red-shift for caffeine possibly linked to a steric interaction absent in theophylline. Free rotation about the O⋯Na+⋯O bond is found to greatly decrease the complexity of the IRMPD spectra of the SCBDs of caffeine and theophylline and demonstrates excellent agreement between the IRMPD and calculated spectra. Electronic structure calculations have been done at the MP2(full)/aug-cc-pCVTZ/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) level of theory using the aug-cc-pCVTZ basis set for Na+ and all Na+-interacting heterotatoms, and the 6-311+G(2d,2p) basis set for all non-interacting atoms within the SCBDs, in order to provide accurate electronic energies. Currently, installation and implementation of a pulsed electrospray high pressure ion source mated to an existing high pressure mass spectrometer (HPMS) is underway. The new ion source will greatly increase the range of possibilities for the study of ion-molecule reactions in the McMahon laboratory. One of the unique features of the new design is the incorporation of a gas-tight electrospray interface, allowing for more possibilities than only the study of cluster-ion equilibria involving hydration (H2On⋯S+), where S+ is an ion produced by electrospray. Other small prototypical biological molecules such as amines and thiols can be used without concern for the toxicity of these species. Another unique design feature allows electrosprayed ions to associate with neutral solvent species in an electric field free reaction chamber (RC). This ensures that values of equilibrium constants determined are truly representative of ions in states of thermochemical equilibrium. The existing HPMS in the McMahon laboratory is limited to the study of small volatile organic molecules. The new ion source will permit the exploration of systems involving non-volatile species, doubly charged ions and many biologically relevant molecules such as amino acids, peptides, nucleobases and carbohydrates.