MS Analysis of a Dilution Series of Bacteria: Phytoplankton to Improve Detection of Low Abundance Bacterial Peptides

Assigning links between microbial activity and biogeochemical cycles in the ocean is a primary objective for ecologists and oceanographers. Bacteria represent a small ecosystem component by mass, but act as the nexus for both nutrient transformation and organic matter recycling. There are limited methods to explore the full suite of active bacterial proteins largely responsible for degradation. Mass spectrometry (MS)-based proteomics now has the potential to document bacterial physiology within these complex systems. Global proteome profiling using MS, known as data dependent acquisition (DDA), is limited by the stochastic nature of ion selection, decreasing the detection of low abundance peptides. The suitability of MS-based proteomics methods in revealing bacterial signatures outnumbered by phytoplankton proteins was explored using a dilution series of pure bacteria (Ruegeria pomeroyi) and diatoms (Thalassiosira pseudonana). Two common acquisition strategies were utilized: DDA and selected reaction monitoring (SRM). SRM improved detection of bacterial peptides at low bacterial cellular abundance that were undetectable with DDA from a wide range of physiological processes (e.g. amino acid synthesis, lipid metabolism, and transport). We demonstrate the benefits and drawbacks of two different proteomic approaches for investigating species-specific physiological processes across relative abundances of bacteria that vary by orders of magnitude

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

MetaGOmics: A Web-Based Tool for Peptide-Centric Functional and Taxonomic Analysis of Metaproteomics Data

Metaproteomics is the characterization of all proteins being expressed by a community of organisms in a complex biological sample at a single point in time. Applications of metaproteomics range from the comparative analysis of environmental samples (such as ocean water and soil) to microbiome data from multicellular organisms (such as the human gut). Metaproteomics research is often focused on the quantitative functional makeup of the metaproteome and which organisms are making those proteins. That is: What are the functions of the currently expressed proteins? How much of the metaproteome is associated with those functions? And, which microorganisms are expressing the proteins that perform those functions? However, traditional protein-centric functional analysis is greatly complicated by the large size, redundancy, and lack of biological annotations for the protein sequences in the database used to search the data. To help address these issues, we have developed an algorithm and web application (dubbed MetaGOmics ) that automates the quantitative functional (using Gene Ontology) and taxonomic analysis of metaproteomics data and subsequent visualization of the results. MetaGOmics is designed to overcome the shortcomings of traditional proteomics analysis when used with metaproteomics data. It is easy to use, requires minimal input, and fully automates most steps of the analysis-including comparing the functional makeup between samples

An Alignment-Free Metapeptide Strategy for Metaproteomic Characterization of Microbiome Samples Using Shotgun Metagenomic Sequencing

In principle, tandem mass spectrometry can be used to detect and quantify the peptides present in a microbiome sample, enabling functional and taxonomic insight into microbiome metabolic activity. However, the phylogenetic diversity constituting a particular microbiome is often unknown, and many of the organisms present may not have assembled genomes. In ocean microbiome samples, with particularly diverse and uncultured bacterial communities, it is difficult to construct protein databases that contain the bulk of the peptides in the sample without losing detection sensitivity due to the overwhelming number of candidate peptides for each tandem mass spectrum. We describe a method for deriving metapeptides (short amino acid sequences that may be represented in multiple organisms) from shotgun metagenomic sequencing of microbiome samples. In two ocean microbiome samples, we constructed site-specific metapeptide databases to detect more than one and a half times as many peptides as by searching against predicted genes from an assembled metagenome and roughly three times as many peptides as by searching against the NCBI environmental proteome database. The increased peptide yield has the potential to enrich the taxonomic and functional characterization of sample metaproteomes

Variations in copepod proteome and respiration rate in association with diel vertical migration and circadian cycle

Author Posting. © University of Chicago, 2018. This article is posted here by permission of University of Chicago for personal use, not for redistribution. The definitive version was published in Biological Bulletin 235 (2018): 30-42, doi:10.1086/699219.The diel vertical migration of zooplankton is a process during which individuals spend the night in surface waters and retreat to depth during the daytime, with substantial implications for carbon transport and the ecology of midwater ecosystems. The physiological consequences of this daily pattern have, however, been poorly studied beyond investigations of speed and the energetic cost of swimming. Many other processes are likely influenced, such as fuel use, energetic trade-offs, underlying diel (circadian) rhythms, and antioxidant responses. Using a new reference transcriptome, proteomic analyses were applied to compare the physiological state of a migratory copepod, Pleuromamma xiphias, immediately after arriving to the surface at night and six hours later. Oxygen consumption was monitored semi-continuously to explore underlying cyclical patterns in metabolic rate under dark-dark conditions. The proteomic analysis suggests a distinct shift in physiology that reflects migratory exertion and changes in metabolism. These proteomic analyses are supported by the respiration experiments, which show an underlying cycle in metabolic rate, with a peak at dawn. This project generates molecular tools (transcriptome and proteome) that will allow for more detailed understanding of the underlying physiological processes that influence and are influenced by diel vertical migration. Further, these studies suggest that P. xiphias is a tractable model for continuing investigations of circadian and diel vertical migration influences on plankton physiology. Previous studies did not account for this cyclic pattern of respiration and may therefore have unrepresented respiratory carbon fluxes from copepods by about 24%.Funding for ET-S was provided by a Training Grant from the National Institutes of Health (T32 HG00035), and proteomics work was supported in part by the University of Washington’s Proteomics Resource (UWPR95794). Funding was provided by Simon’s Foundation International as part of the BIOSSCOPE project.2019-08-1

Metaproteomics Reveal That Rapid Perturbations in Organic Matter Prioritize Functional Restructuring Over Taxonomy In Western Arctic Ocean Microbiomes

We examined metaproteome profiles from two Arctic microbiomes during 10-day shipboard incubations to directly track early functional and taxonomic responses to a simulated algal bloom and an oligotrophic control. Using a novel peptide-based enrichment analysis, significant changes (p-value \u3c 0.01) in biological and molecular functions associated with carbon and nitrogen recycling were observed. Within the first day under both organic matter conditions, Bering Strait surface microbiomes increased protein synthesis, carbohydrate degradation, and cellular redox processes while decreasing C1 metabolism. Taxonomic assignments revealed that the core microbiome collectively responded to algal substrates by assimilating carbon before select taxa utilize and metabolize nitrogen intracellularly. Incubations of Chukchi Sea bottom water microbiomes showed similar, but delayed functional responses to identical treatments. Although 24 functional terms were shared between experimental treatments, the timing, and degree of the remaining responses were highly variable, showing that organic matter perturbation directs community functionality prior to alterations to the taxonomic distribution at the microbiome class level. The dynamic responses of these two oceanic microbial communities have important implications for timing and magnitude of responses to organic perturbations within the Arctic Ocean and how community-level functions may forecast biogeochemical gradients in oceans

The Effects of Ocean Acidification on Multiple Life History Stages of the Pacific Oyster, Crassostrea gigas: Implications for Physiological Trade-offs

Thesis (Ph.D.)--University of Washington, 2014As global climate change accelerates, due in large part to increasing emissions of carbon dioxide and other greenhouse gases from fossil fuel use, agriculture, and large-scale changes in land use, natural ecosystems bear the consequences. For marine systems these include increased mean seawater temperature, changes in carbonate chemistry equilibria, and increased pollutant loading due to non-point run-off, among other effects. Human-induced environmental changes will not have the same magnitude of effect in all regions, but on average the changes occurring are rapid and significant. Natural populations will either need to acclimatize and/or adapt, or shift their ranges to enable continued existence. This dissertation explores the effects of ocean acidification on the Pacific oyster, Crassostrea gigas. Oysters are sedentary and inhabit a naturally variable environment (the intertidal zone) and thus may be pre-adapted to withstand rapid environmental change. Oysters and similarly sedentary organisms are ideal for investigating the effects of environmental change on biology because they are not able to escape these changes, but must respond physiologically (acclimatize) if they are to survive. Due to this ecological history, oysters provide a model that allows us to explore potential physiological mechanisms that are needed in a response to specific environmental changes as well as the limits of these mechanisms. In the first chapter, the effects of elevated partial pressure of CO2 (pCO2, a major driver of ocean acidification) on oyster larvae are explored. Larvae were exposed to low pH during early development, a period that included the transition from energetic dependence on maternally derived lipids to dependence on exogenous resources. Larvae were found to experience a developmental delay at elevated pCO2, manifested as smaller size and slower rate of shell deposition. These significant effects of ocean acidification on early larval development may indicate a bottleneck in the oyster life cycle as the pH of marine waters decreases. Subsequent research has shown that these effects at early larval stages can carry over into later stages after settlement in another oyster species (Hettinger et al. 2012). In order to better understand the effects of environmental change on oyster physiology, we developed proteomic tools to explore changes in protein pathways in oyster gill (ctenidia) tissue. The second chapter explores the gill proteome (suite of expressed proteins) of adult oysters. Characterization of the proteome provides insight into the physiological mechanisms that may be available to the oyster during response to an environmental stress. The results revealed that the ctenidia proteome includes a diverse array of proteins that accomplish many functions and that it is a metabolically active tissue. The proteome sequencing lays the groundwork for exploring how ocean acidification affects various proteomic pathways in the tissue that acts as the interface between the oyster and its environment. Lastly, the adult oyster response to ocean acidification and a second stress are explored via proteomics, fatty acid profiles, glycogen content, shell microstructure, and mortality in response to heat shock. There was a significant impact of ocean acidification on oyster shell integrity, but no effects after one month of exposure on relative amounts of fatty acid, glycogen or response to acute heat shock. Through the proteomic analysis, we revealed an active and significant proteomic response to ocean acidification exposure, uncovering some of the mechanisms behind the observed macro-phenotypic changes. Additionally, the proteomic response to mechanical stimulation was largely altered between low and high pCO2, suggesting that ocean acidification can fundamentally change how oysters respond to a second stress. Works Cited Hettinger, A., Sanford, E., Hill, T.M., Russell, A.D., Sato, K.N., Hoey, J., Forsch, M., Page, H.N. and Gaylord, B. (2012). Persistent carry-over effects of planktonic exposure to ocean acidification in the Olympia oyster. Ecology , 93(12): 2758-2768

Data accompanying Timmins-Schiffman et al. 2013

<p>These 3 files are the source data for Table 1 and Figures 3, 4, and 5 in Timmins-Schiffman et al. 2013 Elevated pCO2 causes developmental delay in early larval Pacific oysters, Crassostrea gigas, published in Marine Biology (doi 10.1007/s00227-012-2055-x).</p> <p>Table 1: Salinity, total alkalinity (AT), and spectrophotometric (spec) pH are point measurements taken each day. Partial pressure of CO2, carbonate saturation, and carbonate ion concentration were calculated from spec pH and AT. Mean and standard deviation (u ± SD) for the following parameters are given for all 3 days: temperature, salinity, AT, pH, pCO2, and carbonate ion concentration.</p> <p>Data for Fig 3: Number of larvae scored as calcified, uncalcified, or partially calcified are given for different replicates (jars) for the two time points (24 and 72 hours post-fertilization) and different pCO2 conditions (400, 700, or 1000 µatm).</p> <p>Data for Figs 4 and 5: Each cell represents a measurement for an individual larva.  Column headers are formatted [measurement type][pCO2].[day measurement taken], i.e. height400.day1 contains data for larval shell height from the 400 uatm treatment on day 1 post-fertilization.  Data are for shell height and depth, pCO2 of approximately 400, 700, and 1000 uatm, and days 1 and 3 post-fertilization.</p> <p>Data for Fig 6: Data were plotted for individual oysters with measurements of both shell height and hinge length.  Columns have data for day post-fertilization, pCO2 (in uatm), DayTreatment combined factor, hinge length, and shell height.  Each row contains data for a single oyster larva.</p

Ocean Acidification Affects the Oyster Proteome

<p>This fileset includes data demonstrating the effects of exposure to high pCO2 (low pH) on global protein expression (the proteome) in Pacific oysters.  Included are the expression data (from Skyline) for each protein averaged within treatment groups (high and low pCO2) with SwissProt ID annotations; a blastp result file associating oyster protein IDs (CGI #s) with KEGG pathway IDs; the input file for iPath to show differential expression between low and high pCO2; and 2 iPath outputs showing pathways identified in the entire oyster proteome as well as pathways that are differentially expressed in the gill at high and low pCO2.  Below is a link to the prezi that adds further explanation.</p