Search for: All records

Peptides and proteins were identified during a controlled laboratory degradation of the marine diatom Thalassiosira weissflogii by a surface seawater microbiome. Samples from each time point were processed both with and without the protease trypsin, allowing a partial differentiation between peptides produced naturally by microbial enzymatic degradation and peptides produced from the laboratory digestion of intact protein. Over the 12-day degradation experiment, 31% of the particulate organic carbon was depleted, and there was no preferential degradation of the overall protein pool. However, there was distinct differentiation in the cellular location, secondary structure and modifications between peptides produced by microbial vs. laboratory breakdown. During the initial period of rapid algal decay and bacterial growth, intracellular components from the cytoplasm were consumed first, resulting in the accumulation of membrane-associated proteins and peptides in the detrital pool. Accompanying the enrichment of membrane protein material was an increase in the importance of ɑ-helix motifs. Methylated arginine, a post-translational modification common in cell senescence, was found in high amounts within the microbially produced detrital peptide pool, suggesting a link between in-cell modification and resistance to immediate degradation. Another modification—asparagine deamidation—accumulated within the detrital peptides. Protein taxonomies showed the bacterial community decomposing the algal material was rich in Proteobacteria, and protein annotations showed abundant transportation of solubilized carbohydrates and small peptides across membranes. At this early stage of diagenesis, no changes in bulk amino acids (THAA) were observed, yet a proteomic approach allowed us to observe selective changes in diatom protein preservation by using amino acid sequences to infer subcellular location, secondary structures, and post-translational modifications (PTMs). 

Abstract. Metaproteomics is an increasingly popular methodology that provides information regarding the metabolic functions of specific microbial taxa and has potential for contributing to ocean ecology and biogeochemical studies. A blinded multi-laboratory intercomparison was conducted to assess comparability and reproducibility of taxonomic and functional results and their sensitivity to methodological variables. Euphotic zone samples from the Bermuda Atlantic Time-Series Study in the North Atlantic Ocean collected by in situ pumps and the AUV Clio were distributed with a paired metagenome, and one-dimensional liquid chromatographic data dependent acquisition mass spectrometry analyses was stipulated. Analysis of mass spectra from seven laboratories through a common informatic pipeline identified a shared set of 1056 proteins from 1395 shared peptides constituents. Quantitative analyses showed good reproducibility: pairwise regressions of spectral counts between laboratories yielded R2 values ranging from 0.43 to 0.83, and a Sørensen similarity analysis of the top 1,000 proteins revealed 70–80 % similarity between laboratory groups. Taxonomic and functional assignments showed good coherence between technical replicates and different laboratories. An informatic intercomparison study, involving 10 laboratories using 8 software packages successfully identified thousands of peptides within the complex metaproteomic datasets, demonstrating the utility of these software tools for ocean metaproteomic research. Future efforts could examine reproducibility in deeper metaproteomes, examine accuracy in targeted absolute quantitation analyses, and develop standards for data output formats to improve data interoperability. Together, these results demonstrate the reproducibility of metaproteomic analyses and their suitability for microbial oceanography research including integration into global scale ocean surveys and ocean biogeochemical models. 

Abstract. Metaproteomics is an increasingly popular methodology that provides information regarding the metabolic functions of specific microbial taxa and has potential for contributing to ocean ecology and biogeochemical studies. A blinded multi-laboratory intercomparison was conducted to assess comparability and reproducibility of taxonomic and functional results and their sensitivity to methodological variables. Euphotic zone samples from the Bermuda Atlantic Time-series Study (BATS) in the North Atlantic Ocean collected by in situ pumps and the autonomous underwater vehicle (AUV) Clio were distributed with a paired metagenome, and one-dimensional (1D) liquid chromatographic data-dependent acquisition mass spectrometry analysis was stipulated. Analysis of mass spectra from seven laboratories through a common bioinformatic pipeline identified a shared set of 1056 proteins from 1395 shared peptide constituents. Quantitative analyses showed good reproducibility: pairwise regressions of spectral counts between laboratories yielded R2 values averaged 0.62±0.11, and a Sørensen similarity analysis of the top 1000 proteins revealed 70 %–80 % similarity between laboratory groups. Taxonomic and functional assignments showed good coherence between technical replicates and different laboratories. A bioinformatic intercomparison study, involving 10 laboratories using eight software packages, successfully identified thousands of peptides within the complex metaproteomic datasets, demonstrating the utility of these software tools for ocean metaproteomic research. Lessons learned and potential improvements in methods were described. Future efforts could examine reproducibility in deeper metaproteomes, examine accuracy in targeted absolute quantitation analyses, and develop standards for data output formats to improve data interoperability. Together, these results demonstrate the reproducibility of metaproteomic analyses and their suitability for microbial oceanography research, including integration into global-scale ocean surveys and ocean biogeochemical models. 

Abstract Models and observations suggest that particle flux attenuation is lower across the mesopelagic zone of anoxic environments compared to oxic environments. Flux attenuation is controlled by microbial metabolism as well as aggregation and disaggregation by zooplankton, all of which shape the relative abundance of differently sized particles. Observing and modeling particle spectra can provide information about the contributions of these processes. We measured particle size spectrum profiles at one station in the oligotrophic Eastern Tropical North Pacific Oxygen Deficient Zone (ETNP ODZ) using an underwater vision profiler (UVP), a high‐resolution camera that counts and sizes particles. Measurements were taken at different times of day, over the course of a week. Comparing these data to particle flux measurements from sediment traps collected over the same time‐period allowed us to constrain the particle size to flux relationship, and to generate highly resolved depth and time estimates of particle flux rates. We found that particle flux attenuated very little throughout the anoxic water column, and at some time points appeared to increase. Comparing our observations to model predictions suggested that particles of all sizes remineralize more slowly in the ODZ than in oxic waters, and that large particles disaggregate into smaller particles, primarily between the base of the photic zone and 500 m. Acoustic measurements of multiple size classes of organisms suggested that many organisms migrated, during the day, to the region with high particle disaggregation. Our data suggest that diel‐migrating organisms both actively transport biomass and disaggregate particles in the ODZ core.