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Dissolved proteins are important components of high molecular weight marine dissolved organic matter (DOM). Little is known about the sources, transformations, and pathways of marine DOM, and <30% has been characterized at the molecular-level. Advances in proteomics technology have improved our ability to sequence proteins, making dissolved proteins a suitable target for molecular-level characterization. Dissolved proteins are important biomarkers in the oceans, and molecular-level analysis can provide valuable insight into their chemical characteristics, structural modifications, and potential sources. Molecular-level characterization of dissolved proteins in seawater requires a reproducible recovery method with efficient recovery yields and minimal sample losses. The use of surface blocking agents (SBA) to saturate sites of non-specific interactions reduced adsorptive sample losses during recovery of dissolved proteins from artificial seawater by tangential flow ultrafiltration. The SBA pre-treatment resulted in more reproducible recovery yields (∼55–60%) and enhanced initial recovery by 46%. Dissolved proteins recovered from deep and surface seawater samples were separated by gel electrophoresis, revealing the presence of a small number of discrete bands and significant amounts of unresolved background. A proteinase K assay determined that the bands and unresolved background were proteinaceous in nature. Enzymatic digests of the dissolved proteins were analyzed by reversed phase chromatography coupled on-line to a linear trapping quadrupole Fourier transform mass spectrometer (LTQ-FTMS). The peptide MS/MS spectra were searched against non-redundant databases by correlation algorithms, but no positive identifications were made. The peptide tandem mass spectra were directly interpreted by de novo sequencing to generate peptide tags that were used in database searches to develop protein homologue information. The similarity search results revealed two trends for specific classes of proteins observed (membrane/envelope proteins and enzymes) and identified conserved sequences for several multi-species protein homologues. These results demonstrate that there are several different classes of refractory proteins represented in the DOM pool and provide evidence for the presence of dissolved proteins other than bacterial outer membrane proteins. Additionally, an intact protein was extracted from a gel band and analyzed by direct infusion ESI FTMS. The resultant high resolution protein spectrum was deconvoluted to determine the accurate molecular weight of the protein.