#METABOLOMICS WORKBENCH TruxalCarlson_20200527_155406 DATATRACK_ID:2022 STUDY_ID:ST001409 ANALYSIS_ID:AN002357 PROJECT_ID:PR000965 VERSION 1 CREATED_ON June 26, 2020, 3:44 pm #PROJECT PR:PROJECT_TITLE Patterns in metabolite pools show that phytoplankton leave a taxon-specific PR:PROJECT_TITLE signature on particulate carbon: North Pacific Transition Zone depth profile PR:PROJECT_TYPE Marine Metabolomics PR:PROJECT_SUMMARY In the surface ocean, carbon is fixed by phytoplankton and respired by the PR:PROJECT_SUMMARY entire marine community at an astonishingly high rate. At any point in time, the PR:PROJECT_SUMMARY difference between these two processes yields a carbon pool in surface particles PR:PROJECT_SUMMARY that is a combination of both freshly fixed and partially degraded material. On PR:PROJECT_SUMMARY a molecular level, we have a limited knowledge of the small molecules, or PR:PROJECT_SUMMARY metabolites, within this pool. Specific metabolites have been shown to be PR:PROJECT_SUMMARY responsible for fueling respiration, maintaining organismal interactions, and PR:PROJECT_SUMMARY transferring energy throughout the microbial community. Metabolomics, or the PR:PROJECT_SUMMARY direct observation and quantification of the small molecules that are the result PR:PROJECT_SUMMARY of cellular activity, provides an important lens through which we can begin to PR:PROJECT_SUMMARY assess the standing stocks of small compounds that likely fuel a great deal of PR:PROJECT_SUMMARY heterotrophic activity in the surface ocean. Here we describe community PR:PROJECT_SUMMARY metabolomes of particulate material into the North Pacific Ocean and compare the PR:PROJECT_SUMMARY metabolomes to a variety of phytoplankton grown in the lab. Using both targeted PR:PROJECT_SUMMARY and untargeted metabolomics, we identify metabolites in the particulate carbon PR:PROJECT_SUMMARY pool and explore their latitudinal and phylogenetic distributions. This analysis PR:PROJECT_SUMMARY reveals several compounds that have not been previously recognized as abundant PR:PROJECT_SUMMARY components of the marine organic carbon pool. We found that the community PR:PROJECT_SUMMARY metabolome showed distinct differences between the regimes that likely reflects PR:PROJECT_SUMMARY the phytoplankton community present. The community metabolome in surface waters PR:PROJECT_SUMMARY of the subtropical domain was remarkably consistent even when sampled weeks PR:PROJECT_SUMMARY apart, while the northern regions showed a patchier and less reproducible PR:PROJECT_SUMMARY community metabolome. Some individual compounds showed distinct patterns between PR:PROJECT_SUMMARY oceanographic regimes, including homarine, an abundant molecule that can PR:PROJECT_SUMMARY contribute up to 4% of the total particulate carbon pool in marine surface PR:PROJECT_SUMMARY waters. Glutamic acid and glutamine showed opposite patterns in the PR:PROJECT_SUMMARY oceanographic regimes, suggesting differences in community-level nitrogen PR:PROJECT_SUMMARY assimilation in these different regimes. Overall, this study offers a new PR:PROJECT_SUMMARY perspective into particulate carbon composition in oceanographic research, PR:PROJECT_SUMMARY reveals important carbon pools that may fuel the microbial loop, and suggests an PR:PROJECT_SUMMARY altered community-level nitrogen assimilation capacity over the North Pacific PR:PROJECT_SUMMARY transition zone. PR:INSTITUTE University of Washington PR:DEPARTMENT School of Oceanography PR:LABORATORY Ingalls Lab PR:LAST_NAME Heal PR:FIRST_NAME Katherine PR:ADDRESS 1501 NE Boat Street, Marine Science Building, Room G, Seattle, WA, 98195, USA PR:EMAIL kheal@uw.edu PR:PHONE 206-616-4658 #STUDY ST:STUDY_TITLE Patterns in metabolite pools show that phytoplankton leave a taxon-specific ST:STUDY_TITLE signature on particulate carbon: North Pacific Transition Zone depth profile ST:STUDY_TYPE Marine metabolomics depth profile ST:STUDY_SUMMARY In the surface ocean, carbon is fixed by phytoplankton and respired by the ST:STUDY_SUMMARY entire marine community at an astonishingly high rate. At any point in time, the ST:STUDY_SUMMARY difference between these two processes yields a carbon pool in surface particles ST:STUDY_SUMMARY that is a combination of both freshly fixed and partially degraded material. On ST:STUDY_SUMMARY a molecular level, we have a limited knowledge of the small molecules, or ST:STUDY_SUMMARY metabolites, within this pool. Specific metabolites have been shown to be ST:STUDY_SUMMARY responsible for fueling respiration, maintaining organismal interactions, and ST:STUDY_SUMMARY transferring energy throughout the microbial community. Metabolomics, or the ST:STUDY_SUMMARY direct observation and quantification of the small molecules that are the result ST:STUDY_SUMMARY of cellular activity, provides an important lens through which we can begin to ST:STUDY_SUMMARY assess the standing stocks of small compounds that likely fuel a great deal of ST:STUDY_SUMMARY heterotrophic activity in the surface ocean. Here we describe community ST:STUDY_SUMMARY metabolomes of particulate material into the North Pacific Ocean and compare the ST:STUDY_SUMMARY metabolomes to a variety of phytoplankton grown in the lab. Using both targeted ST:STUDY_SUMMARY and untargeted metabolomics, we identify metabolites in the particulate carbon ST:STUDY_SUMMARY pool and explore their latitudinal and phylogenetic distributions. This analysis ST:STUDY_SUMMARY reveals several compounds that have not been previously recognized as abundant ST:STUDY_SUMMARY components of the marine organic carbon pool. We found that the community ST:STUDY_SUMMARY metabolome showed distinct differences between the regimes that likely reflects ST:STUDY_SUMMARY the phytoplankton community present. The community metabolome in surface waters ST:STUDY_SUMMARY of the subtropical domain was remarkably consistent even when sampled weeks ST:STUDY_SUMMARY apart, while the northern regions showed a patchier and less reproducible ST:STUDY_SUMMARY community metabolome. Some individual compounds showed distinct patterns between ST:STUDY_SUMMARY oceanographic regimes, including homarine, an abundant molecule that can ST:STUDY_SUMMARY contribute up to 4% of the total particulate carbon pool in marine surface ST:STUDY_SUMMARY waters. Glutamic acid and glutamine showed opposite patterns in the ST:STUDY_SUMMARY oceanographic regimes, suggesting differences in community-level nitrogen ST:STUDY_SUMMARY assimilation in these different regimes. Overall, this study offers a new ST:STUDY_SUMMARY perspective into particulate carbon composition in oceanographic research, ST:STUDY_SUMMARY reveals important carbon pools that may fuel the microbial loop, and suggests an ST:STUDY_SUMMARY altered community-level nitrogen assimilation capacity over the North Pacific ST:STUDY_SUMMARY transition zone. ST:INSTITUTE University of Washington ST:DEPARTMENT School of Oceanography ST:LABORATORY Ingalls Lab ST:LAST_NAME Heal ST:FIRST_NAME Katherine ST:ADDRESS 1501 NE Boat Street ST:EMAIL kheal@uw.edu ST:PHONE 206-616-4658 #SUBJECT SU:SUBJECT_TYPE Other SU:SUBJECT_SPECIES Natural mixed marine microbial community SU:GENDER Not applicable #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Blk0p2_1 Depth_m:NA | Vol_L:NA Latitude=NA; Longitude=NA; UTC=NA; Replicate=1; Type=Blk; RAW_FILE_NAME=180702_Blk_Blk0p2_1;180702_Blk_Blk0p2_1;180716_Blk_Blk0p2_1 SUBJECT_SAMPLE_FACTORS - S7E41D30_1 Depth_m:30 | Vol_L:4 Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D30_1;180702_Smp_S7E41D30_1;180716_Smp_S7E41D30_1 SUBJECT_SAMPLE_FACTORS - S7E41D40_1 Depth_m:40 | Vol_L:4 Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D40_1;180702_Smp_S7E41D40_1;180716_Smp_S7E41D40_1 SUBJECT_SAMPLE_FACTORS - S7E41D60_1 Depth_m:60 | Vol_L:4 Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D60_1;180702_Smp_S7E41D60_1;180716_Smp_S7E41D60_1 SUBJECT_SAMPLE_FACTORS - S7E41D80_1 Depth_m:80 | Vol_L:4 Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D80_1;180702_Smp_S7E41D80_1;180716_Smp_S7E41D80_1 SUBJECT_SAMPLE_FACTORS - S7E41D100_1 Depth_m:100 | Vol_L:4 Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D100_1;180702_Smp_S7E41D100_1;180716_Smp_S7E41D100_1 SUBJECT_SAMPLE_FACTORS - S7E41D120_1 Depth_m:120 | Vol_L:4 Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D120_1;180702_Smp_S7E41D120_1;180716_Smp_S7E41D120_1 SUBJECT_SAMPLE_FACTORS - S7E41D140_1 Depth_m:140 | Vol_L:4 Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D140_1;180702_Smp_S7E41D140_1;180716_Smp_S7E41D140_1 SUBJECT_SAMPLE_FACTORS - S7E41D160_1 Depth_m:160 | Vol_L:4 Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D160_1;180702_Smp_S7E41D160_1;180716_Smp_S7E41D160_1 SUBJECT_SAMPLE_FACTORS - S7E41D180_1 Depth_m:180 | Vol_L:4 Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D180_1;180702_Smp_S7E41D180_1;180716_Smp_S7E41D180_1 SUBJECT_SAMPLE_FACTORS - S7E41D250_1 Depth_m:250 | Vol_L:4 Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D250_1;180702_Smp_S7E41D250_1;180716_Smp_S7E41D250_1 SUBJECT_SAMPLE_FACTORS - TruePoo_Half1 Depth_m:NA | Vol_L:NA Latitude=NA; Longitude=NA; UTC=NA; Replicate=1; Type=Pool; RAW_FILE_NAME=180702_Poo_TruePoo_Half1;180702_Poo_TruePoo_Half1;180716_Poo_TruePoo10_Half1 SUBJECT_SAMPLE_FACTORS - TruePoo_Half2 Depth_m:NA | Vol_L:NA Latitude=NA; Longitude=NA; UTC=NA; Replicate=2; Type=Pool; RAW_FILE_NAME=180702_Poo_TruePoo_Half2;180702_Poo_TruePoo_Half2;180726_Poo_TruePoo10_Half2 SUBJECT_SAMPLE_FACTORS - TruePoo_Half3 Depth_m:NA | Vol_L:NA Latitude=NA; Longitude=NA; UTC=NA; Replicate=3; Type=Pool; RAW_FILE_NAME=180702_Poo_TruePoo_Half3;180702_Poo_TruePoo_Half3;180716_Poo_TruePoo10_Half3 SUBJECT_SAMPLE_FACTORS - TruePoo_Full1 Depth_m:NA | Vol_L:NA Latitude=NA; Longitude=NA; UTC=NA; Replicate=1; Type=Pool; RAW_FILE_NAME=180702_Poo_TruePoo_Full1;180702_Poo_TruePoo_Full1;180716_Poo_TruePoo10_Full1 SUBJECT_SAMPLE_FACTORS - TruePoo_Full2 Depth_m:NA | Vol_L:NA Latitude=NA; Longitude=NA; UTC=NA; Replicate=2; Type=Pool; RAW_FILE_NAME=180702_Poo_TruePoo_Full2;180702_Poo_TruePoo_Full2;180716_Poo_TruePoo10_Full2 SUBJECT_SAMPLE_FACTORS - TruePoo_Full3 Depth_m:NA | Vol_L:NA Latitude=NA; Longitude=NA; UTC=NA; Replicate=3; Type=Pool; RAW_FILE_NAME=180702_Poo_TruePoo_Full3;180702_Poo_TruePoo_Full3;180716_Poo_TruePoo10_Full3 #COLLECTION CO:COLLECTION_SUMMARY Samples for particulate metabolites were collected from 10 depths between 30 and CO:COLLECTION_SUMMARY 250 m on June 3, 2017 aboard the R/V Marcus Langseth. At each sampling location CO:COLLECTION_SUMMARY and depth, single, duplicate, or triplicate filters were collected using either CO:COLLECTION_SUMMARY niskins attached to a conductivity, temperature, depth array (CTD) or the CO:COLLECTION_SUMMARY underway intake. Samples (4 L) were collected into polycarbonate carboys, CO:COLLECTION_SUMMARY filtered onto 147 mm 0.2 μm PTFE filters using peristaltic pumps, polycarbonate CO:COLLECTION_SUMMARY filter holders, and Masterflex PharMed BPT tubing (Cole-Parmer). Filters were CO:COLLECTION_SUMMARY flash frozen in liquid nitrogen and stored at -80°C until extraction. In CO:COLLECTION_SUMMARY addition to our samples, we filtered seawater through two 0.2 μm PTFE filters CO:COLLECTION_SUMMARY in series, using the second filter as a methodological blank. This blank is CO:COLLECTION_SUMMARY especially important to parse signals from the salt matrix that would be present CO:COLLECTION_SUMMARY after sorption to the filter. CO:SAMPLE_TYPE Suspended Marine Particulate Matter CO:STORAGE_CONDITIONS Described in summary #TREATMENT TR:TREATMENT_SUMMARY No treatment, this is a study of the natural marine microbial population in the TR:TREATMENT_SUMMARY North Pacific Transition Zone. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Each sample was extracted using a modified Bligh-Dyer extraction. Briefly, SP:SAMPLEPREP_SUMMARY filters were cut up and put into 15 mL teflon centrifuge tubes containing a SP:SAMPLEPREP_SUMMARY mixture of 100 µm and 400 µm silica beads. Heavy isotope-labeled internal SP:SAMPLEPREP_SUMMARY standards were added along with ~2 mL of cold aqueous solvent (50:50 SP:SAMPLEPREP_SUMMARY methanol:water) and ~3 mL of cold organic solvent (dichloromethane). The samples SP:SAMPLEPREP_SUMMARY were shaken on a FastPrep-24 Homogenizer for 30 seconds and chilled in a -20 °C SP:SAMPLEPREP_SUMMARY freezer repeatedly for three cycles of bead-beating and a total of 30 minutes of SP:SAMPLEPREP_SUMMARY chilling. The organic and aqueous layers were separated by spinning samples in a SP:SAMPLEPREP_SUMMARY centrifuge at 4,300 rpm for 2 minutes at 4 °C. The aqueous layer was removed to SP:SAMPLEPREP_SUMMARY a new glass centrifuge tube. The remaining organic fraction was rinsed three SP:SAMPLEPREP_SUMMARY more times with additions of 1 to 2 mL of 50:50 methanol:water. All aqueous SP:SAMPLEPREP_SUMMARY rinses were combined for each sample and dried down under N2 gas. The remaining SP:SAMPLEPREP_SUMMARY organic layer was transferred into a clean glass centrifuge tube and the SP:SAMPLEPREP_SUMMARY remaining bead beating tube was rinsed two more times with cold organic solvent. SP:SAMPLEPREP_SUMMARY The combined organic rinses were centrifuged, transferred to a new tube, and SP:SAMPLEPREP_SUMMARY dried under N2 gas. Dried aqueous fractions were re-dissolved in 380 µL of SP:SAMPLEPREP_SUMMARY water. Dried organic fractions were re-dissolved in 380 µL of 1:1 SP:SAMPLEPREP_SUMMARY water:acetonitrile. 20 µL of isotope-labeled injection standards in water were SP:SAMPLEPREP_SUMMARY added to both fractions. Blank filters were extracted alongside samples as SP:SAMPLEPREP_SUMMARY methodological blanks. SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACTION_METHOD Bligh-Dyer SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY See attached summary. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity I-Class CH:COLUMN_NAME Waters Acquity UPLC HSS Cyano (100 x 2.1mm, 1.8um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS See attached protocol. MS:MS_RESULTS_FILE ST001409_AN002357_Results.txt UNITS:Adjusted and normalized peak areas Has m/z:Yes Has RT:Yes RT units:Seconds #END