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Small biomolecule systems were interrogated using infrared multiple photon dissociation (IRMPD) action spectroscopy and corresponding quantum chemical calculations with a particular focus on peptide fragmentation and protonation site preference. b2+ and b3+ fragment ions with a terminal lysine homolog residue were investigated using IRMPD in the fingerprint region (1000 cm-1 – 2000 cm-1) and a variety of computational methods. We present the first spectroscopic confirmation of b-ion formation with a lactam structure. Infrared spectra for b2+ fragment system indicate the presence of a mixture of structures, though final determination will require further investigation. The b3+ fragment ion infrared spectra show strong support for the presence of a predominantly lactam structure. Extensive computational research in this system suggests the B3LYP method to be the most computationally efficient density functional theory method for spectral predictions. However, the inclusion of p-polarization into the basis set yielded inconclusive results and should be investigated further.

Complex organic mixtures in the environment can contain hundreds to thousands of different organic molecules, and their composition and reactivity can have important environmental implications. In addition to gases, the atmosphere is made of a variety of small liquids and solids called aerosols. These aerosols have large impacts on human health, climate, and atmospheric chemical reactions. Here, secondary organic aerosol (SOA) from the ozonolysis of α-pinene is characterized. The atmospheric lifetime of SOA is very uncertain, but recent laboratory and modeling studies have demonstrated that photolysis is potentially an important process for organic mass loss from aerosol particles.1-5 Photolysis modifies the molecular composition and properties of aerosols through photolytic cleaving and repartitioning of volatile products. Characterization of dry, irradiated SOA can provide insights into photolysis driven changes in absorption properties and chemical composition. These results illuminate aging mechanisms and chemical and physical properties of organic aerosols in order to improve atmospheric modeling and the understanding of atmospheric chemical reactions. However, the high chemical complexity and low atmospheric abundance presents a difficult analytical challenge. Milligrams, or more, of material may be needed for speciated spectroscopic analysis.6 This study used a suite of advanced analytical techniques, including a novel combination of action spectroscopy and mass spectrometry that provides more structural information on organic mixtures than mass spectrometry alone. This study also used tunable light from a free electron laser, infrared and UV/Vis absorption, and computational chemistry to characterize molecules in α-pinene SOA. In addition, complex organic mixtures are also found in particulate matter that has deposited onto Earth’s surface. The preliminary results of dew analysis, including a foundation method of analysis for future study, gives the first look at organic material deposited into dew water on natural surfaces. This offers insight into atmospheric organic deposition to better understand chemical transport, air quality, and carbon cycling in the atmosphere.

Naturally occurring humic substances are a combination of decayed plant and soil materials that have been incorporated into a complex matrix with other organic substrates. These acidic humic substances account for a portion of the organic matter found in soil and in the soluble organic portion of fresh water. These humic acids, which are polar and straw-colored, are derived from soil humus and terrestrial and aquatic plants. Considerable attention has been given to these naturally occurring humic materials since the chlorination of these acids in our drinking water results in the formation of trihalomethanes and other chloro-organic compounds. Many of these chlorinated compounds have been determined by the standard Ames Salmonela test to be mutagenic. Despite extensive research concerning humic materials, their structure, and the environmental impact of the derived chlorinated organics are still not fully understood. This research involved an extensive analytical study on a local water source, Forge Pond, which is located in Granby, Massachusetts. The pond water was collected, chlorinated, and analyzed by gas chromatography with electron capture detection to determine the levels of chloro-organics formed when the pH, chlorine dose, and reaction time were varied. It was determined that these variables are critical in the formation of chlorinated organic products. Water was also collected from this pond and the humic material extracted by adsorption and concentration on XAD-8 resin. The thermal properties of the humic materials was studied by Thermal Gravimetric Analysis, and Pyrolysis/Gas Chromatography-Mass Spectrometry was used for chemical characterization. In general, humic acid showed prominent signals related to polysaccharides and phenolic derivatives whereas fulvic acid showed more polysaccharides, and less phenolic signals. A commercial humic acid showed analytical composition mainly related to benzene derivatives.

Newly developed flow field-flow fractionation-inductively coupled plasma mass spectrometry (FlFFF-ICP-MS) has been explored for its capability of size-based elemental speciation. Information obtained from this combination technique includes size and molecular weight distributions, diffusion coefficient, polydispersity, and molecular conformation. Various biological, environmental, and industrial samples are examined to illustrate the versatility of FlFFF-ICP-MS. For biological applications complexation of metals (Co, Cr, Fe, La, Th, and U) with three important proteins (albumin, ferritin, and transferrin) is examined. Size characterization of elements (Al, Cu, Pb, and Zn) in food macromolecules also is investigated. For environmental applications elemental size characterization of Al, Cu, Pb and Zn in humic acids is examined. Physical parameters such as diffusion coefficient and hydrodynamic diameter are determined. Furthermore, FlFFF is employed to study the aggregation of humic acid in the presence of alkaline earth ions (Ca, Sr, and Ba). An equivalent sphere density index (DESI) is proposed and used to trace the conformational arrangement of humic macromolecules. In addition, the FlFFF-ICP-MS is used for elemental size characterization of river and estuarine sediment core samples and aquatic colloids. To demonstrate the scope of the FlFFF-ICP-MS technique with industrial materials, size characterization of Al, Fe, Pb, Si, Ti, and Zr in various chemical mechanical polishing slurries is examined. Particle size information, including size distribution, minimum and maximum particle sizes, average and mean diameters, polydispersity, and breadth of distribution are obtained. Owing to the similarity of FlFFF and a membrane filtration technique, a preliminary investigation of applying a FlFFF channel as an on-channel matrix removal and analyte preconcentration before ICP-MS detection is carried out. Various analyte elements are approximately 50-fold preconcentrated and matrix-removed from 5,000 mg l−1 Ca and Na salt solutions. Frit outlet preconcentration after FlFFF size separation also is examined. With a frit outlet, a standard cross flow nebulizer does not maintain the added benefit from the FlFFF frit outlet. Therefore, use of micronebulizer is necessary for ICP-MS detection and is evaluated in this study.

A mass spectrometry-based method was developed for studying hemoglobin assembly/dissociation. The dynamics of bovine hemoglobin assembly were investigated by monitoring monomers/oligomers equilibria in solution with electrospray ionization mass spectrometry and circular dichroism spectroscopy. The mass spectral data confirm that bovine hemoglobin dissociation involves a step where heme is first lost from the β-chain of the α*β*-dimer to form a heme-deficient dimeric species or semi-hemoglobin dimer (α*β*). The experimental data provide strong evidence that binding of a partially unstructured apo-β-chain to a tightly folded holo-α-chain to form the semi-hemoglobin dimer is the initial step of hemoglobin assembly. Such binding locks the β-chain in a highly ordered conformation, which allows for an efficient heme acquisition. This step is followed by the docking of the two hemoglobin dimers to form a tetrameric form of the protein. Also, investigation of the individual apo- and heme-reconstituted globin chains show that although apo-α- and apo-β-chains are very flexible, dynamic, and have similar protein structures (the globin fold), reconstituted α-chains are monomeric and exhibit conformational structures very similar to holo-α-chains in the complex Hb equilibrium mixture. Reconstituted β-chains oligomerize to form the tetrameric homo-β-globin species HbH found in alpha thalassemic disorder. This suggests that dynamics play an important, perhaps the most important, role in the hemoglobin assembly process. We hypothesize that the intrinsic protein disorder exhibited by the bovine hemoglobin β-chains and the asymmetry of globin interaction reported in this work developed due to evolutionary pressure to provide a vital safeguard that may inhibit random oligomerization and aggregation in the crowded environment of the red blood cell, thus directing the assembly process along the correct pathway. The data support this hypothesis as hemoglobins analyzed from all organisms that evolved after the emergence of separate α- and β-chains (bovine, porcine, human, and modern fish hemoglobins) exhibit the expected asymmetry in the roles played by their globin chains in assembly. In contrast, hemoglobin from the mollusk Scapharca, which parallels the emergence of separate α- and β-chains does not exhibit asymmetry. In the analysis of the modern fish hemoglobin from Gadus morhua, the Atlantic cod, it was noticed that the measured molecular weights for the α- and β-chains did not correspond to masses calculated from their published sequences. Using mass spectrometry-based methods 65% sequence coverage was achieved, and a number of mutations were identified in both globin chains. A feasibility study for the use of mass spectrometry to study the interaction between hemoglobin and its scavenger protein haptoglobin is also presented.

Protein ion charge state distributions in Electrospray ionization mass spectrometry (ESI MS) provide information on protein dynamics, as it contains contributions from all protein conformers present in solution. Such ionic contributions often overlap, limiting the amount of useful information that can be extracted from the spectra. This difficulty is overcome by using factor analysis, which allows spectral deconvolution to be carried out and information on individual protein conformers to be extracted. Experiments were carried out by acquiring a series of spectra over a range of near-native and denaturing conditions and then subjecting to singular value decomposition, to determine the number of conformers. Ionic contributions of each conformer to the total signal are then determined using a supervised minimization routine. By monitoring acid and alcohol induced equilibrium intermediates of well-characterized model proteins we can demonstrate that factor analysis correctly predicts the total number of conformations of the model proteins under a variety of conditions. Several factors have also been suggested as determinants of the number of charges displayed by the protein ions in electrospray ionization (ESI) spectra, such as available basic residues, solvent composition, etc. This work demonstrates that protein surface area in solution is the major determinant of the number of charges accommodated by a protein ion in the gas phase. The charged residue model of ESI process predicts that the number of charges (N) accommodated by a globular protein in the gas phase should be related to its surface area (S) as N∼S3/4. We analyzed a set of proteins ranging from 5 kDa insulin to 0.5 MDa, ferritin under native conditions. The average charge of each protein was plotted as a function of its surface area (calculated based on the available crystal structures), giving an apparent correlation of N∼S0.68. Effects of gas phase chemistry on the charge state distribution was investigated. It is seen that using a stronger base as a electrolyte component resulted in a noticeable decrease of the average charge, without affecting the correlation N∼S 0.68. At high pH charge state distribution is shifted to lower charges, attributed to the free base in solution.

Monitoring hydrogen-deuterium exchange of proteins can yield a wealth of information about not only the native state, but also not native states of proteins. These non-native states have important biological roles including protein folding intermediates as well as ligand binding/delivery and protein-protein interactions. Structural and dynamic properties of a partially folded conformation (A-state) of ubiquitin are studied using amide hydrogen exchange in solution (HDX) and mass spectrometric detection. A clear distinction between the native state of the protein and the A-state can be made when HDX is carried out in a semicorrelated regime. Furthermore, combination of HDX and protein ion fragmentation in the gas phase by means of collision-induced dissociation (CAD)] is used to evaluate the conformational stability of various protein segments specifically in the molten globular state. Chain flexibility appears to be distributed very unevenly in this non-native conformation. This study also demonstrates the power of mass spectrometry as a tool in providing conformer-specific information about the structure and dynamics of both native and non-native protein states coexisting in solution under equilibrium. This dissertation has been broken down into several subsections. First, we evaluate electrospray ionization amide hydrogen exchange collision assisted dissociation mass spectrometry's (ESI HDX CAD MS) methodology to better understand the determinants of hydrogen scrambling in the gas phase, which can be used to probe non-native states of proteins. Secondly we examined the structure of a molten globule of ubiquitin using HDX CAD MS under mildly denaturing conditions and compare this with the proposed NMR and crystal structures of the A-state and native state, respectively. Lastly, we have conducted studies include ESI MS studies of non-covalent interactions between cellular retinoic acid binding protein II (CRABP II) and the ligand-binding domain (LBD) of retinoic acid receptor (RAR) as it pertains to delivery of retinoic acid (RA). The backbone dynamics of CRABP II will be investigated in order better understand the ligand binding and delivery properties of CRABP II and how they differ from CRABP I.

The selectivity and sensitivity offered by the combination of liquid chromatography (LC) and mass spectrometry (MS) make it a powerful tool for studying the chemistry of compounds that are of environmental and biological importance. This dissertation focuses on developing LC-MS methods to study metal-interacting molecules in marine and biological systems. In the studies of marine systems, organic ligands that bind Cu(II) in estuarine waters and molecules that are produced by the archaeon Pyrobaculum aerophilum to reduce Fe(III) to Fe(II) have been investigated. Two groups of Cu(II)-binding ligands in the Chesapeake Bay have been isolated by immobilized-metal affinity chromatography (IMAC). Reversed-phase liquid chromatography (RPLC) analyses indicate that both groups of ligands are hydrophilic in nature. Further MS analyses indicate that at least one of the ligands is made up of sulfur- and nitrogen-containing functional groups. LC-MS has also been used to isolate and characterize molecules that are produced by the archaeon Pyrobaculum aerophilum to reduce Fe(III) to Fe(II). Further characterization of these compounds by UV-Vis spectroscopy, NMR spectroscopy, and tandem MS (i.e. MS/MS) indicates that at least one of these compounds contains amide and quinone-like groups. In the studies of biological systems, the gentle nature of electrospray ionization (ESI)-MS has been used to study metal-protein and protein-protein complexes of β-2-microglobulin (β2m), which is the protein component of the amyloid fibers that cause dialysis-related amyloidosis. MS, size-exclusion chromatography (SEC), and dynamic light scattering (DLS) analyses indicate that in the presence of Cu(II) β2m forms amyloid fibers by the building up of dimeric units. Furthermore, MS, SEC, and DLS data suggest that the hexamer is the nucleus that is required for the formation of the amyloid fibers. Taken as a whole, MS, SEC, DLS, and X-ray fluorescence data also suggest that Cu(II) is necessary to reach the hexameric state, but Cu(II) is released upon formation of the mature fibers. Finally, ESI-MS has been used to estimate the surface areas of β2m oligomers. Our surface area measurements suggest that the hexamer has a more compact structure than the dimer or tetramer, suggesting that Cu is released by the tetramer upon formation of the hexamer.