Development of chromatographic and mass spectrometric tools to study metal-interacting molecules in environmental and biological systems .

摘要

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 beta-2-microglobulin (beta2m), 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) beta2m 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 beta2m 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.