Phytoplankton Plastid Proteomics: Cracking Open Diatoms to Understand Plastid Biochemistry Under Iron Limitation

Diatoms, such as Thalassiosira pseudonana, are important oceanic primary producers, as they sequester carbon dioxide (CO₂) out of the atmosphere, die, and precipitate to the ocean floor. In many areas of the world’s oceans, phytoplankton, such as diatoms, are limited in growth by the availability of iron (Fe). Fe is an essential nutrient for phytoplankton, as it is central in the electron transport chain component of photosynthesis. Through this study, we examined if Fe-limitation makes a significant difference in the proteins expressed within the chloroplast, the power source for diatoms. Here, we utilized a new plastid isolation technique specific to diatoms and completed 14 mass spectrometry experiments to determine how many proteins transit the plastid membrane, if there are any differences in the expressed proteomes within the plastid grown under Fe-replete and Fe-limited conditions, and what those differences are. Over 900 unique proteins were identified from the isolated plastids, and cluster analyses of the data verified that statistical differences are present between the Fe-replete and Fe-limited growth conditions. Furthermore, our plastid proteome is in agreement with many of the recognized proteins previously discovered in land plant plastids, suggesting the isolation method followed by proteomic mass spectrometry is valid and sensitive. Through the isolation and analysis of plastid proteins, as shown here, scientists can now better identify which nutrients and/or trace metals directly affect diatom photosynthetic capacity so they can design better experiments to increase CO₂ fixation rates

Identifying and Tracking Proteins Through the Marine Water Column: Insights Into the Inputs and Preservation Mechanisms of Protein in Sediments

Proteins generated during primary production represent an important fraction of marine organic nitrogen and carbon, and have the potential to provide organism-specific information in the environment. The Bering Sea is a highly productive system dominated by seasonal blooms and was used as a model system for algal proteins to be tracked through the water column and incorporated into detrital sedimentary material. Samples of suspended and sinking particles were collected at multiple depths along with surface sediments on the continental shelf and deeper basin of the Bering Sea. Modified standard proteomic preparations were used in conjunction with high pressure liquid chromatography-tandem mass spectrometry to identify the suite of proteins present and monitor changes in their distribution. In surface waters 207 proteins were identified, decreasing through the water column to 52 proteins identified in post-bloom shelf surface sediments and 24 proteins in deeper (3490 m) basin sediments. The vast majority of identified proteins in all samples were diatom in origin, reflecting their dominant contribution of biomass during the spring bloom. Identified proteins were predominantly from metabolic, binding/structural, and transport-related protein groups. Significant linear correlations were observed between the number of proteins identified and the concentration of total hydrolysable amino acids normalized to carbon and nitrogen. Organelle-bound, transmembrane, photosynthetic, and other proteins involved in light harvesting were preferentially retained during recycling. These findings suggest that organelle and membrane protection represent important mechanisms that enhance the preservation of protein during transport and incorporation into 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

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

The Path to Preservation: Using Proteomics to Decipher the Fate of Diatom Proteins During Microbial Degradation

We drew upon recent advances in tandem mass spectrometry-based proteomic analyses in order to examine the proteins that remain after a diatom bloom enters the stationary phase, precipitates out of the photic zone, and is subjected to microbial degradation over a 23-d period within a controlled laboratory environment. Proteins were identified from tandem mass spectra searched against three different protein databases in order to track proteins from Thalassiosira pseudonana and any potential bacterial contributions. A rapid loss of diatom protein was observed over the incubation period; 75% of the proteins initially identified were not detected after 72 h of exposure to a microbial population. By the 23rd day, peptides identified with high confidence correlated with only four T. pseudonana proteins. Five factors may have influenced the preservation of diatom proteins: (1) protection within organelles or structures with multiple membranes, (2) the relative cellular abundance in the photosynthetic apparatus, (3) the number of transmembrane domains in the protein sequence, (4) the presence of glycan modification motifs, and (5) the capability of proteins or peptides to aggregate into supramolecules

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