#METABOLOMICS WORKBENCH TruxalCarlson_20200428_130830 DATATRACK_ID:1996 STUDY_ID:ST001372 ANALYSIS_ID:AN002291 PROJECT_ID:PR000938 VERSION 1 CREATED_ON May 5, 2020, 1:21 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 Subtropical Gyre 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 patichier 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 612-616-4840 #STUDY ST:STUDY_TITLE Patterns in metabolite pools show that phytoplankton leave a taxon-specific ST:STUDY_TITLE signature on particulate carbon: North Pacific Subtropical Gyre 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 patichier 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, Marine Science Building, Room G ST:EMAIL kheal@uw.edu ST:PHONE 612-616-4840 #SUBJECT SU:SUBJECT_TYPE Other SU:SUBJECT_SPECIES Natural mixed marine microbial community #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 - FilterA Depth_m:NA | Vol_L:NA Latitude=NA; Longitude=NA; UTC=NA; Replicate=A; Type=Blk; RAW_FILE_NAME=170410_Blk_FilterBlk_A;170410_Blk_FilterBlk_A;170413_Blk_FilterBlk_A SUBJECT_SAMPLE_FACTORS - FilterB Depth_m:NA | Vol_L:NA Latitude=NA; Longitude=NA; UTC=NA; Replicate=B; Type=Blk; RAW_FILE_NAME=170410_Blk_FilterBlk_B;170410_Blk_FilterBlk_B;170413_Blk_FilterBlk_B SUBJECT_SAMPLE_FACTORS - KM1513-15m_A Depth_m:15 | Vol_L:10.7 Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=A; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-15m_A;170410_Smp_KM1513-15m_A;170413_Smp_KM1513-15m_A SUBJECT_SAMPLE_FACTORS - KM1513-15m_B Depth_m:15 | Vol_L:10.7 Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=B; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-15m_B;170410_Smp_KM1513-15m_B;170413_Smp_KM1513-15m_B SUBJECT_SAMPLE_FACTORS - KM1513-15m_C Depth_m:15 | Vol_L:10.7 Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=C; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-15m_C;170410_Smp_KM1513-15m_C;170413_Smp_KM1513-15m_C SUBJECT_SAMPLE_FACTORS - KM1513-45m_A Depth_m:45 | Vol_L:9 Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=A; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-45m_A;170410_Smp_KM1513-45m_A;170413_Smp_KM1513-45m_A SUBJECT_SAMPLE_FACTORS - KM1513-45m_B Depth_m:45 | Vol_L:9 Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=B; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-45m_B;170410_Smp_KM1513-45m_B;170413_Smp_KM1513-45m_B SUBJECT_SAMPLE_FACTORS - KM1513-45m_C Depth_m:45 | Vol_L:9 Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=C; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-45m_C;170410_Smp_KM1513-45m_C;170413_Smp_KM1513-45m_C SUBJECT_SAMPLE_FACTORS - KM1513-75m_A Depth_m:75 | Vol_L:12.7 Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=A; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-75m_A;170410_Smp_KM1513-75m_A;170413_Smp_KM1513-75m_A SUBJECT_SAMPLE_FACTORS - KM1513-75m_B Depth_m:75 | Vol_L:12.7 Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=B; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-75m_B;170410_Smp_KM1513-75m_B;170413_Smp_KM1513-75m_B SUBJECT_SAMPLE_FACTORS - KM1513-75m_C Depth_m:75 | Vol_L:12.7 Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=C; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-75m_C;170410_Smp_KM1513-75m_C;170413_Smp_KM1513-75m_C SUBJECT_SAMPLE_FACTORS - KM1513-125m_A Depth_m:125 | Vol_L:12.17 Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=A; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-125m_A;170410_Smp_KM1513-125m_A;170413_Smp_KM1513-125m_A SUBJECT_SAMPLE_FACTORS - KM1513-125m_B Depth_m:125 | Vol_L:12.17 Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=B; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-125m_B;170410_Smp_KM1513-125m_B;170413_Smp_KM1513-125m_B SUBJECT_SAMPLE_FACTORS - KM1513-125m_C Depth_m:125 | Vol_L:12.17 Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=C; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-125m_C;170410_Smp_KM1513-125m_C;170413_Smp_KM1513-125m_C SUBJECT_SAMPLE_FACTORS - April11AqExtractsHalf_1 Depth_m:NA | Vol_L:NA Latitude=NA; Longitude=NA; UTC=NA; Replicate=1; Type=Pool; RAW_FILE_NAME=170410_Poo_April11AqExtractsHalf_1;170410_Poo_April11AqExtractsHalf_1;170413_Poo_April11AqExtractsHalf_1 SUBJECT_SAMPLE_FACTORS - April11AqExtractsHalf_2 Depth_m:NA | Vol_L:NA Latitude=NA; Longitude=NA; UTC=NA; Replicate=2; Type=Pool; RAW_FILE_NAME=170410_Poo_April11AqExtractsHalf_2;170410_Poo_April11AqExtractsHalf_2;170413_Poo_April11AqExtractsHalf_2 SUBJECT_SAMPLE_FACTORS - April11AqExtractsHalf_3 Depth_m:NA | Vol_L:NA Latitude=NA; Longitude=NA; UTC=NA; Replicate=3; Type=Pool; RAW_FILE_NAME=170410_Poo_April11AqExtractsHalf_3;170410_Poo_April11AqExtractsHalf_3;170413_Poo_April11AqExtractsHalf_3 SUBJECT_SAMPLE_FACTORS - April11AqExtractsFull_1 Depth_m:NA | Vol_L:NA Latitude=NA; Longitude=NA; UTC=NA; Replicate=1; Type=Pool; RAW_FILE_NAME=170410_Poo_April11AqExtractsFull_1;170410_Poo_April11AqExtractsFull_1;170413_Poo_April11AqExtractsFull_1 SUBJECT_SAMPLE_FACTORS - April11AqExtractsFull_2 Depth_m:NA | Vol_L:NA Latitude=NA; Longitude=NA; UTC=NA; Replicate=2; Type=Pool; RAW_FILE_NAME=170410_Poo_April11AqExtractsFull_2;170410_Poo_April11AqExtractsFull_2;170413_Poo_April11AqExtractsFull_2 SUBJECT_SAMPLE_FACTORS - April11AqExtractsFull_3 Depth_m:NA | Vol_L:NA Latitude=NA; Longitude=NA; UTC=NA; Replicate=3; Type=Pool; RAW_FILE_NAME=170410_Poo_April11AqExtractsFull_3;170410_Poo_April11AqExtractsFull_3;170413_Poo_April11AqExtractsFull_3 #COLLECTION CO:COLLECTION_SUMMARY Samples for particulate metabolites were collected from different water depths CO:COLLECTION_SUMMARY by niskin bottles attached to a conductivity, temperature, depth array (CTD). CO:COLLECTION_SUMMARY Metabolite samples were filtered onto 142 mm 0.2 µm Durapore filters using CO:COLLECTION_SUMMARY peristaltic, polycarbonate filter holders, and Masterflex PharMed BPT tubing CO:COLLECTION_SUMMARY (Cole-Parmer). Filters were quenched in liquid nitrogen immediately after CO:COLLECTION_SUMMARY filtration and stored at -80°C until extraction. Each sample was 30-40 L CO:COLLECTION_SUMMARY filtered seawater; each filter was split into three equal parts for triplicate CO:COLLECTION_SUMMARY extractions. A blank PTFE filter was extracted alongside samples as a CO:COLLECTION_SUMMARY methodological blank. CO:SAMPLE_TYPE Suspended Marine Particulate Matter CO:STORAGE_CONDITIONS Described in summary #TREATMENT TR:TREATMENT_SUMMARY No treatment - this was a study of the natural marine microbial population at TR:TREATMENT_SUMMARY different depths in the North Pacific Subtropical Gyre. #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 HILIC CH:INSTRUMENT_NAME Waters Acquity I-Class CH:COLUMN_NAME SeQuant ZIC- pHILIC (150 x 2.1mm, 5um) #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 NEGATIVE MS:MS_COMMENTS See attached protocol MS:MS_RESULTS_FILE ST001372_AN002291_Results.txt UNITS:Adjusted and normalized peak areas Has m/z:Yes Has RT:Yes RT units:Seconds #END