A Proteomics Approach to the Examination of Proteins in Marine Systems
The response of global carbon and nitrogen cycles to future climate change is uncertain. In order to understand the impacts that future changes to climate will have on these cycles, a more detailed understanding of them is essential. This dissertation utilizes a combined approach of molecular biomarkers and proteomic investigations to elucidate historic source material contributions and microbial protein production to contribute to a more thorough understanding of the marine carbon and nitrogen cycles. The examination of molecular organic biomarkers throughout an Arctic sediment core showed the dominant input in the area was from marine sources with lower but steady contributions from terrestrial sources during the Holocene. Attempts to recover proteins from deeper sediments to correlate with lipid biomarkers were unsuccessful but led to the optimization of an extraction protocol for an added protein standard, bovine serum albumin, from sediments. An investigation into the expressed proteome of the heterotrophic marine bacterium, Ruegeria pomeroyi, under environmentally realistic carbon supply conditions during exponential and stationary growth phases identified over 2000 proteins. The most abundant proteins identified were responsible for porins, transport, binding, translation, and protein refolding and could represent potential biomarkers of bacterial processes and/or activity. A parallel study of R. pomeroyi, in which 13C-labeled leucine was added to the culture during exponential growth phase, showed labeled incorporation ranging from 16 to 21% of the total proteins produced depending on growth phase. The widespread distribution of the label among the growth phases indicates active recycling by the bacteria. This study demonstrates a method through which bacterial protein synthesis can be tracked. A study of the marine diatom Thalassiosira pseudonana acclimated to iron replete or iron-limited conditions showed iron-limited organisms increased proteins involved in pathways associated with intracellular protein recycling, the pentose phosphate pathway, lower photosynthetic energy production, enhancement of photorespiration, and increased polysaccharide production. This application of proteomics to the examination of proteins in marine sediments, a marine diatom, and a heterotrophic marine bacterium shows the potential for these techniques to help elucidate the fate of proteins in marine environments and could be used in conjunction with well-established molecular organic marker studies.
Sample Processing Protocol
"Determining whether past climatic or environmental changes can be determined by identifying changes to organic matter source inputs throughout a sediment core was investigated with a multi-proxy approach that included molecular organic markers (fatty acid, alkane, alcohol and sterol analysis), a novel proxy, the Branched and Isoprenoid Tetraethers (BIT) index (a Glycerol Dialkyl Glycerol Tetraether lipid analysis), and bulk C and stable isotope measurements. These measures were used to examine sediments from 18 sampling horizons within the core. R. pomeroyi was cultured under environmentally realistic C conditions and samples were collected at four time points throughout its growth phases representing early and late exponential phase and early and late stationary phase. Digested peptides within the samples were separated prior to introduction into the mass spectrometer through reverse-phase chromatography which utilized an analytical column (20 cm long, 75 μm id fused silica capillary column packed with C18 particles (Magic C18AQ, 100 Å, 5 μm; Michrom, Bioresources) preceded by a pre-column (2 cm long, 100 μm (Magic C18AQ, 200 Å, 5 μm; Michrom). Peptides were eluted using an acidified (formic acid, 0.1% v/v) water-acetonitrile gradient (5 to 35% acetonitrile in 90 min). Tandem mass spectrometry (LC-MS/MS) of the peptides was performed on a QExactive mass-spectrometer (Thermo Fisher) and on a LTQ-Orbitrap hybrid mass- spectrometer (Thermo Fisher) for the blanks."
Data Processing Protocol
Mass spectral results were interpreted and searched with SEQUEST (v. 2012.01.0) (Eng et al., 2008) to match observed spectra to theoretical spectra generated from the predicted peptide sequences determined by the search database chosen. All CID spectra were searched against the most recent version of the R. pomeroyi proteome (ASM1196v2; ncbi.nlm.nih.gov/genome; 4,278 proteins) and 50 common protein contaminants in the lab (e.g. keratins, trypsin). The searches were conducted with enzyme specificity for trypsin and a +6 Da modification specific to leucine residues. The alteration to the leucine was searched because these samples were run on the MS at the same time as another study using a 13C-leucine label and the potential for carry-over between samples needed to be taken into consideration. Match probability between the 94 observed and theoretical spectra was set at 90% (p<0.1) on ProteinProphet and PetptideProphet (Keller et al., 2002). Proteins with only one identified peptide were excluded.