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

Electrophoretic extraction and proteomic characterization of proteins buried in marine sediments

Proteins are the largest defined molecular component of marine organic nitrogen, and hydrolysable amino acids, the building blocks of proteins, are important components of particulate nitrogen in marine sediments. In oceanic systems, the largest contributors are phytoplankton proteins, which have been tracked from newly produced bloom material through the water column to surface sediments in the Bering Sea, but it is not known if proteins buried deeper in sediment systems can be identified with confidence. Electrophoretic gel protein extraction methods followed by proteomic mass spectrometry and database searching were used as the methodology to identify buried phytoplankton proteins in sediments from the 8-10 cm section of a Bering Sea sediment core. More peptides and proteins were identified using an SDS-PAGE tube gel than a standard 1D flat gel or digesting the sediment directly with trypsin. The majority of proteins identified correlated to the marine diatom, Thalassiosira pseudonana, rather than bacterial protein sequences, indicating an algal source not only dominates the input, but also the preserved protein fraction. Abundant RuBisCO and fucoxanthin chlorophyll a/c binding proteins were identified, supporting algal sources of these proteins and reinforcing the proposed mechanisms that might protect proteins for long time periods. Some preserved peptides were identified in unexpected gel molecular weight ranges, indicating that some structural changes or charge alteration influenced the mobility of these products during electrophoresis isolation. Identifying buried photosystem proteins suggests that algal particulate matter is a significant fraction of the preserved organic carbon and nitrogen pools in marine sediments

Diatom Proteomics Reveals Unique Acclimation Strategies to Mitigate Fe Limitation

Phytoplankton growth rates are limited by the supply of iron (Fe) in approximately one third of the open ocean, with major implications for carbon dioxide sequestration and carbon (C) biogeochemistry. To date, understanding how alteration of Fe supply changes phytoplankton physiology has focused on traditional metrics such as growth rate, elemental composition, and biophysical measurements such as photosynthetic competence (Fv/Fm). Researchers have subsequently employed transcriptomics to probe relationships between changes in Fe supply and phytoplankton physiology. Recently, studies have investigated longer-term (i.e. following acclimation) responses of phytoplankton to various Fe conditions. In the present study, the coastal diatom, Thalassiosira pseudonana, was acclimated (10 generations) to either low or high Fe conditions, i.e. Fe-limiting and Fe-replete. Quantitative proteomics and a newly developed proteomic profiling technique that identifies low abundance proteins were employed to examine the full complement of expressed proteins and consequently the metabolic pathways utilized by the diatom under the two Fe conditions. A total of 1850 proteins were confidently identified, nearly tripling previous identifications made from differential expression in diatoms. Given sufficient time to acclimate to Fe limitation, T. pseudonana up-regulates proteins involved in pathways associated with intracellular protein recycling, thereby decreasing dependence on extracellular nitrogen (N), C and Fe. The relative increase in the abundance of photorespiration and pentose phosphate pathway proteins reveal novel metabolic shifts, which create substrates that could support other well-established physiological responses, such as heavily silicified frustules observed for Fe-limited diatoms. Here, we discovered that proteins and hence pathways observed to be down-regulated in short-term Fe starvation studies are constitutively expressed when T. pseudonana is acclimated (i.e., nitrate and nitrite transporters, Photosystem II and Photosystem I complexes). Acclimation of the diatom to the desired Fe conditions and the comprehensive proteomic approach provides a more robust interpretation of this dynamic proteome than previous studies.This work was supported by National Science Foundation grants OCE1233014 (BLN) and the Office of Polar Programs Postdoctoral Fellowship grant 0444148 (BLN). DRG was supported by National Institutes of Health 5P30ES007033-10. AH and MTM were supported by Natural Sciences and Engineering Research Council of Canada. RFS and PWB were supported by the New Zealand Royal Society Marsden Fund and the Ministry of Science. This work is supported in part by the University of Washington's Proteomics Computer Resource Centre (UWPR95794). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Protein Recycling in Bering Sea Algal Incubations

Protein present in phytoplankton represents a large fraction of the organic nitrogen and carbon transported from its synthesis in surface waters to marine sediments. Yet relatively little is known about the longevity of identifiable protein in situ, or the potential modifications to proteins that occur during bloom termination, protein recycling and degradation. To address this knowledge gap, diatom-dominated phytoplankton was collected during the Bering Sea spring blooms of 2009 and 2010, and incubated under darkness in separate shipboard degradation experiments spanning 11 and 53 d, respectively. In each experiment, the protein distribution was monited over time using shotgun proteomics, along with total hydrolyzable amino acids (THAAs), total protein, particulate organic carbon (POC) and nitrogen (PN), and bacterial cell abundance. Identifiable proteins, total protein and THAAs were rapidly lost during the first 5 d of enclosure in darkness in both incubations. Thereafter the loss rate was slower, and it declined further after 22 d. The initial loss of identifiable biosynthetic, glycolysis, metabolism and translation proteins after 12 h may represent shutdown of cellular activity among algal cells. Additional peptides with glycan modifications were identified in early incubation time points, suggesting that such protein modifications could be used as a marker for internal recycling processes and possibly cell death. Protein recycling was not uniform, with a subset of algal proteins including fucoxanthin chlorophyll binding proteins and RuBisCO identified after 53 d of degradation. Non-metric multidimensional scaling was used to compare the incubations with previous environmental results. The results confirmed recent observations that some fraction of algal proteins can survive water column recycling and undergo transport to marine sediments, thus contributing organic nitrogen to the benthos

Evaluation of Electrophoretic Protein Extraction and Database-Driven Protein Identification from Marine Sediments

Intact proteins comprise a major component of organic carbon and nitrogen produced globally and are likely an important fraction of organic matter in sediments and soils. Extracting the protein component from sediments and soils for mass spectral characterization and identification represents a substantial challenge given the range of products and functionalities present in the complex matrix. Multiple forms of gel electrophoresis were evaluated as a means of enhancing recovery of sedimentary protein before proteomic characterization and compared with a direct enzymatic digestion of proteins in sediments. Resulting tryptic peptides were analyzed using shotgun proteomics and tandem mass spectra were evaluated with SEQUEST. Multiple databases were then evaluated to examine the ability to confidently identify proteins from environmental samples. Following evaluation of electrophoretic extraction of proteins from sediments, the recovery of an experimentally added standard protein (BSA) from older (\u3e1 ky) sediments was optimized. Protein extraction from sediments via direct electrophoresis of a slurry mixture and the specified extraction buffer resulted in the greatest number of confident protein identifications and highest sequence coverage of the BSA standard. Searching tandem mass spectral data against larger databases with a higher diversity of proteomes did not yield a greater number of, or more confidence in, protein identifications. Regardless of the protein database used, identified peptides correlated to proteins with the same function across taxa. This suggests that while determining taxonomic-level information remains a challenge in samples with unknown mixed species, it is possible to confidently assign the function of the identified protein

Growth Phase Proteomics of the Heterotrophic Marine Bacterium \u3ci\u3eRuegeria pomeroyi\u3c/i\u3e

The heterotrophic marine bacterium, Ruegeria pomeroyi, was experimentally cultured under environmentally realistic carbon conditions and with a tracer-level addition of 13C-labeled leucine to track bacterial protein biosynthesis through growth phases. A combination of methods allowed observation of real-time bacterial protein production to understand metabolic priorities through the different growth phases. Over 2000 proteins were identified in each experimental culture from exponential and stationary growth phases. Within two hours of the 13C-labeled leucine addition, R. pomeroyi significantly assimilated the newly encountered substrate into new proteins. This dataset provides a fundamental baseline for understanding growth phase differences in molecular physiology of a cosmopolitan marine bacterium

Diatom Proteomics Reveals Unique Acclimation Strategies to Mitigate Fe Limitation

Phytoplankton growth rates are limited by the supply of iron (Fe) in approximately one third of the open ocean, with major implications for carbon dioxide sequestration and carbon (C) biogeochemistry. To date, understanding how alteration of Fe supply changes phytoplankton physiology has focused on traditional metrics such as growth rate, elemental composition, and biophysical measurements such as photosynthetic competence (Fv/Fm). Researchers have subsequently employed transcriptomics to probe relationships between changes in Fe supply and phytoplankton physiology. Recently, studies have investigated longer-term (i.e. following acclimation) responses of phytoplankton to various Fe conditions. In the present study, the coastal diatom, Thalassiosira pseudonana, was acclimated (10 generations) to either low or high Fe conditions, i.e. Fe-limiting and Fe-replete. Quantitative proteomics and a newly developed proteomic profiling technique that identifies low abundance proteins were employed to examine the full complement of expressed proteins and consequently the metabolic pathways utilized by the diatom under the two Fe conditions. A total of 1850 proteins were confidently identified, nearly tripling previous identifications made from differential expression in diatoms. Given sufficient time to acclimate to Fe limitation, T. pseudonana up-regulates proteins involved in pathways associated with intracellular protein recycling, thereby decreasing dependence on extracellular nitrogen (N), C and Fe. The relative increase in the abundance of photorespiration and pentose phosphate pathway proteins reveal novel metabolic shifts, which create substrates that could support other well-established physiological responses, such as heavily silicified frustules observed for Fe-limited diatoms. Here, we discovered that proteins and hence pathways observed to be down-regulated in short-term Fe starvation studies are constitutively expressed when T. pseudonana is acclimated (i.e., nitrate and nitrite transporters, Photosystem II and Photosystem I complexes). Acclimation of the diatom to the desired Fe conditions and the comprehensive proteomic approach provides a more robust interpretation of this dynamic proteome than previous studies

Too Big to Fail — U.S. Banks’ Regulatory Alchemy: Converting an Obscure Agency Footnote into an “At Will” Nullification of Dodd-Frank’s Regulation of the Multi-Trillion Dollar Financial Swaps Market

The multi-trillion-dollar market for, what was at that time wholly unregulated, over-the-counter derivatives (“swaps”) is widely viewed as a principal cause of the 2008 worldwide financial meltdown. The Dodd-Frank Act, signed into law on July 21, 2010, was expressly considered by Congress to be a remedy for this troublesome deregulatory problem. The legislation required the swaps market to comply with a host of business conduct and anti-competitive protections, including that the swaps market be fully transparent to U.S. financial regulators, collateralized, and capitalized. The statute also expressly provides that it would cover foreign subsidiaries of big U.S. financial institutions if their swaps trading could adversely impact the U.S. economy or represent the use of extraterritorial trades as an attempt to “evade” Dodd-Frank. In July 2013, the CFTC promulgated an 80-page, triple-columned, and single-spaced “guidance” implementing Dodd-Frank’s extraterritorial reach, i.e., that manner in which Dodd-Frank would apply to swaps transactions executed outside the United States. The key point of that guidance was that swaps trading within the “guaranteed” foreign subsidiaries of U.S. bank holding company swaps dealers were subject to all of Dodd-Frank’s swaps regulations wherever in the world those subsidiaries’ swaps were executed. At that time, the standardized industry swaps agreement contemplated that, inter alia, U.S. bank holding company swaps dealers’ foreign subsidiaries would be “guaranteed” by their corporate parent, as was true since 1992. In August 2013, without notifying the CFTC, the principal U.S. bank holding company swaps dealer trade association privately circulated to its members standard contractual language that would, for the first time, “deguarantee” their foreign subsidiaries. By relying only on the obscure footnote 563 of the CFTC guidance’s 662 footnotes, the trade association assured its swaps dealer members that the newly deguaranteed foreign subsidiaries could (if they so chose) no longer be subject to Dodd-Frank. As a result, it has been reported (and it also has been understood by many experts within the swaps industry) that a substantial portion of the U.S. swaps market has shifted from the large U.S. bank holding companies swaps dealers and their U.S. affiliates to their newly deguaranteed “foreign” subsidiaries, with the attendant claim by these huge big U.S. bank swaps dealers that Dodd-Frank swaps regulation would not apply to these transactions. The CFTC also soon discovered that these huge U.S. bank holding company swaps dealers were “arranging, negotiating, and executing” (“ANE”) these swaps in the United States with U.S. bank personnel and, only after execution in the U.S., were these swaps formally “assigned” to the U.S. banks’ newly “deguaranteed” foreign subsidiaries with the accompanying claim that these swaps, even though executed in the U.S., were not covered by Dodd-Frank. In October 2016, the CFTC proposed a rule that would have closed the “deguarantee” and “ANE” loopholes completely. However, because it usually takes at least a year to finalize a “proposed” rule, this proposed rule closing the loopholes in question was not finalized prior to the inauguration of President Trump. All indications are that it will never be finalized during a Trump Administration. Thus, in the shadow of the recent tenth anniversary of the Lehman failure, there is an understanding among many market regulators and swaps trading experts that large portions of the swaps market have moved from U.S. bank holding company swaps dealers and their U.S. affiliates to their newly deguaranteed foreign affiliates where Dodd- Frank swaps regulation is not being followed. However, what has not moved abroad is the very real obligation of the lender of last resort to rescue these U.S. swaps dealer bank holding companies if they fail because of poorly regulated swaps in their deguaranteed foreign subsidiaries, i.e., the U.S. taxpayer. While relief is unlikely to be forthcoming from the Trump Administration or the Republican-controlled Senate, some other means will have to be found to avert another multi-trillion-dollar bank bailout and/or a financial calamity caused by poorly regulated swaps on the books of big U.S. banks. This paper notes that the relevant statutory framework affords state attorneys general and state financial regulators the right to bring so-called “parens patriae” actions in federal district court to enforce, inter alia, Dodd- Frank on behalf of a state’s citizens. That kind of litigation to enforce the statute’s extraterritorial provisions is now badly needed

Pilot Trial of the Reboot Online Program: An Internet-Delivered, Multidisciplinary Pain Management Program for Chronic Pain

Objectives. Chronic pain causes significant disability and psychological distress, but barriers often prevent people with pain from engaging in traditional face-to-face pain management programs. Accessible, feasible, and effective alternative treatment options are needed. Methods. A prospective, feasibility pilot study was conducted to trial a novel, multidisciplinary online pain management program: the “Reboot Online” program. Twenty participants experiencing pain of at least three months duration were recruited. All participants were enrolled in the “Reboot Online” program, consisting of eight online lessons completed over 16 weeks. Lessons incorporated multidisciplinary input from medical pain specialists, physiotherapists, and psychologists. Participants were assessed at pretreatment, posttreatment, and follow-up using a suite of outcome measures examining pain, disability, catastrophising, self-efficacy, mood, and psychological distress. Results. 13 participants completed the program (65% adherence). Following treatment, the participants had significantly improved scores on measures of pain-related disability, self-efficacy, catastrophising thoughts, acceptance of pain, symptoms of depression, and general psychological distress. These findings were retained at three months posttreatment. Participants also reported high levels of acceptability and satisfaction with the program. Discussion. This study provides pilot evidence for the feasibility, acceptability, and effectiveness of an online, multidisciplinary pain program: “Reboot Online.” Future investigations will focus on conducting a randomised controlled trial of this innovative and promising treatment for chronic pain. This trial is registered with ACTRN12615000660583

Diatom Proteomics Reveals Unique Acclimation Strategies to Mitigate Fe Limitation

Phytoplankton growth rates are limited by the supply of iron (Fe) in approximately one third of the open ocean, with major implications for carbon dioxide sequestration and carbon (C) biogeochemistry. To date, understanding how alteration of Fe supply changes phytoplankton physiology has focused on traditional metrics such as growth rate, elemental composition, and biophysical measurements such as photosynthetic competence (Fv/Fm). Researchers have subsequently employed transcriptomics to probe relationships between changes in Fe supply and phytoplankton physiology. Recently, studies have investigated longer-term (i.e. following acclimation) responses of phytoplankton to various Fe conditions. In the present study, the coastal diatom, Thalassiosira pseudonana, was acclimated (10 generations) to either low or high Fe conditions, i.e. Fe-limiting and Fe-replete. Quantitative proteomics and a newly developed proteomic profiling technique that identifies low abundance proteins were employed to examine the full complement of expressed proteins and consequently the metabolic pathways utilized by the diatom under the two Fe conditions. A total of 1850 proteins were confidently identified, nearly tripling previous identifications made from differential expression in diatoms. Given sufficient time to acclimate to Fe limitation, T. pseudonana up-regulates proteins involved in pathways associated with intracellular protei