#METABOLOMICS WORKBENCH wkumler_20230717_155835 DATATRACK_ID:4170 STUDY_ID:ST002788 ANALYSIS_ID:AN004537 PROJECT_ID:PR001738 VERSION 1 CREATED_ON July 20, 2023, 8:22 am #PROJECT PR:PROJECT_TITLE The influence of mesoscale eddy features on marine metabolomic variability in PR:PROJECT_TITLE the North Pacific Subtropical Gyre PR:PROJECT_TYPE Marine Metabolomics PR:PROJECT_SUMMARY Mesoscale eddies are a dominant source of variability in the ocean's gyres, PR:PROJECT_SUMMARY often analogized to the "weather" of the sea. They alter the balance between PR:PROJECT_SUMMARY light and nutrients, shifting community composition on both the species and PR:PROJECT_SUMMARY molecular scale. Here, we collected metabolomic samples from across eddy dipoles PR:PROJECT_SUMMARY in the North Pacific Subtropical Gyre to detect and quantify these shifts on a PR:PROJECT_SUMMARY chemical level. The data indicate that metabolites dynamically and robustly PR:PROJECT_SUMMARY track with biological community metrics and result in biochemically distinct PR:PROJECT_SUMMARY particulate matter in cyclonic and anticyclonic eddies. PR:INSTITUTE University of Washington PR:DEPARTMENT School of Oceanography PR:LABORATORY Ingalls Lab PR:LAST_NAME Kumler PR:FIRST_NAME William PR:ADDRESS 1501 NE Boat St, Seattle, WA, 98105, USA PR:EMAIL wkumler@uw.edu PR:PHONE 2062216732 PR:FUNDING_SOURCE Simons Collaboration on Ocean Processes and Ecology, NSF #STUDY ST:STUDY_TITLE Metabolomic analysis of particulate matter in the NPSG during a 2018 cruise on ST:STUDY_TITLE the R/V Falkor ST:STUDY_SUMMARY Targeted and untargeted analysis of metabolomics samples from the North Pacific ST:STUDY_SUMMARY Subtropical Gyre taken during the 2018 March/April SCOPE cruise on the R/V ST:STUDY_SUMMARY Falkor (FK180310) across a mesoscale eddy dipole. Particulate matter was ST:STUDY_SUMMARY collected on 0.2um filters and extracted using a modified Bligh & Dyer before ST:STUDY_SUMMARY analysis on a QE Orbitrap. Results show significant changes in the absolute ST:STUDY_SUMMARY quantity and relative composition of particles in the gyre between anticyclonic ST:STUDY_SUMMARY and cyclonic eddies. ST:INSTITUTE University of Washington ST:DEPARTMENT School of Oceanography ST:LABORATORY Ingalls Lab ST:LAST_NAME Kumler ST:FIRST_NAME William ST:ADDRESS 1501 NE Boat St, Seattle, WA 98105 ST:EMAIL wkumler@uw.edu ST:PHONE 2062216732 #SUBJECT SU:SUBJECT_TYPE Other organism 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 190715_Smp_FK180310S77C1-25m_A.mzML 190715_Smp_FK180310S77C1-25m_A.mzML Sample_type:Sample | Station:77 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:20.8044 Time_sampled=2018-04-07 22:57:24; Latitude=24.5306; Longitude=160.787; Temperature=22.8817; Salinity=35.0647; Particulate_carbon=2.1415 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S77C1-25m_B.mzML 190715_Smp_FK180310S77C1-25m_B.mzML Sample_type:Sample | Station:77 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:20.8044 Time_sampled=2018-04-07 22:57:24; Latitude=24.5306; Longitude=160.787; Temperature=22.8817; Salinity=35.0647; Particulate_carbon=2.1415 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S77C1-25m_C.mzML 190715_Smp_FK180310S77C1-25m_C.mzML Sample_type:Sample | Station:77 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:20.8044 Time_sampled=2018-04-07 22:57:24; Latitude=24.5306; Longitude=160.787; Temperature=22.8817; Salinity=35.0647; Particulate_carbon=2.1415 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S77C1-DCM_A.mzML 190715_Smp_FK180310S77C1-DCM_A.mzML Sample_type:Sample | Station:77 | Depth:DCM | Absolute_depth:125 | Sea_level_anomaly:19.87 Time_sampled=2018-04-07 22:57:24; Latitude=24.512; Longitude=160.507; Temperature=22.579; Salinity=35.161; Particulate_carbon=1.48794 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S77C1-DCM_B.mzML 190715_Smp_FK180310S77C1-DCM_B.mzML Sample_type:Sample | Station:77 | Depth:DCM | Absolute_depth:125 | Sea_level_anomaly:19.87 Time_sampled=2018-04-07 22:57:24; Latitude=24.512; Longitude=160.507; Temperature=22.579; Salinity=35.161; Particulate_carbon=1.48794 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S77C1-DCM_C.mzML 190715_Smp_FK180310S77C1-DCM_C.mzML Sample_type:Sample | Station:77 | Depth:DCM | Absolute_depth:125 | Sea_level_anomaly:19.87 Time_sampled=2018-04-07 22:57:24; Latitude=24.512; Longitude=160.507; Temperature=22.579; Salinity=35.161; Particulate_carbon=1.48794 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S80C1-25m_A.mzML 190715_Smp_FK180310S80C1-25m_A.mzML Sample_type:Sample | Station:80 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:20.8044 Time_sampled=2018-04-08 11:14:18; Latitude=24.5306; Longitude=160.787; Temperature=22.8817; Salinity=35.0647; Particulate_carbon=2.1415 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S80C1-25m_B.mzML 190715_Smp_FK180310S80C1-25m_B.mzML Sample_type:Sample | Station:80 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:20.8044 Time_sampled=2018-04-08 11:14:18; Latitude=24.5306; Longitude=160.787; Temperature=22.8817; Salinity=35.0647; Particulate_carbon=2.1415 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S80C1-25m_C.mzML 190715_Smp_FK180310S80C1-25m_C.mzML Sample_type:Sample | Station:80 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:20.8044 Time_sampled=2018-04-08 11:14:18; Latitude=24.5306; Longitude=160.787; Temperature=22.8817; Salinity=35.0647; Particulate_carbon=2.1415 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S80C1-DCM_A.mzML 190715_Smp_FK180310S80C1-DCM_A.mzML Sample_type:Sample | Station:80 | Depth:DCM | Absolute_depth:120 | Sea_level_anomaly:21.03 Time_sampled=2018-04-08 11:14:18; Latitude=24.582; Longitude=160.882; Temperature=22.614; Salinity=35.158; Particulate_carbon=1.59016 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S80C1-DCM_B.mzML 190715_Smp_FK180310S80C1-DCM_B.mzML Sample_type:Sample | Station:80 | Depth:DCM | Absolute_depth:120 | Sea_level_anomaly:21.03 Time_sampled=2018-04-08 11:14:18; Latitude=24.582; Longitude=160.882; Temperature=22.614; Salinity=35.158; Particulate_carbon=1.59016 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S80C1-DCM_C.mzML 190715_Smp_FK180310S80C1-DCM_C.mzML Sample_type:Sample | Station:80 | Depth:DCM | Absolute_depth:120 | Sea_level_anomaly:21.03 Time_sampled=2018-04-08 11:14:18; Latitude=24.582; Longitude=160.882; Temperature=22.614; Salinity=35.158; Particulate_carbon=1.59016 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S62C1-25m_A.mzML 190715_Smp_FK180310S62C1-25m_A.mzML Sample_type:Sample | Station:62 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:-12.48 Time_sampled=2018-04-03 22:32:50; Latitude=22.2318; Longitude=156.855; Temperature=23.0028; Salinity=35.1175; Particulate_carbon=2.35172 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S62C1-25m_B.mzML 190715_Smp_FK180310S62C1-25m_B.mzML Sample_type:Sample | Station:62 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:-12.48 Time_sampled=2018-04-03 22:32:50; Latitude=22.2318; Longitude=156.855; Temperature=23.0028; Salinity=35.1175; Particulate_carbon=2.35172 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S62C1-25m_C.mzML 190715_Smp_FK180310S62C1-25m_C.mzML Sample_type:Sample | Station:62 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:-12.48 Time_sampled=2018-04-03 22:32:50; Latitude=22.2318; Longitude=156.855; Temperature=23.0028; Salinity=35.1175; Particulate_carbon=2.35172 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S62C1-DCM_A.mzML 190715_Smp_FK180310S62C1-DCM_A.mzML Sample_type:Sample | Station:62 | Depth:DCM | Absolute_depth:98 | Sea_level_anomaly:-13.6 Time_sampled=2018-04-03 22:32:50; Latitude=22.367; Longitude=156.57; Temperature=21.264; Salinity=35.176; Particulate_carbon=2.31849 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S62C1-DCM_B.mzML 190715_Smp_FK180310S62C1-DCM_B.mzML Sample_type:Sample | Station:62 | Depth:DCM | Absolute_depth:98 | Sea_level_anomaly:-13.6 Time_sampled=2018-04-03 22:32:50; Latitude=22.367; Longitude=156.57; Temperature=21.264; Salinity=35.176; Particulate_carbon=2.31849 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S62C1-DCM_C.mzML 190715_Smp_FK180310S62C1-DCM_C.mzML Sample_type:Sample | Station:62 | Depth:DCM | Absolute_depth:98 | Sea_level_anomaly:-13.6 Time_sampled=2018-04-03 22:32:50; Latitude=22.367; Longitude=156.57; Temperature=21.264; Salinity=35.176; Particulate_carbon=2.31849 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S64C1-25m_A.mzML 190715_Smp_FK180310S64C1-25m_A.mzML Sample_type:Sample | Station:64 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:-12.48 Time_sampled=2018-04-04 10:50:59; Latitude=22.2318; Longitude=156.855; Temperature=23.0028; Salinity=35.1175; Particulate_carbon=2.35172 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S64C1-25m_B.mzML 190715_Smp_FK180310S64C1-25m_B.mzML Sample_type:Sample | Station:64 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:-12.48 Time_sampled=2018-04-04 10:50:59; Latitude=22.2318; Longitude=156.855; Temperature=23.0028; Salinity=35.1175; Particulate_carbon=2.35172 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S64C1-25m_C.mzML 190715_Smp_FK180310S64C1-25m_C.mzML Sample_type:Sample | Station:64 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:-12.48 Time_sampled=2018-04-04 10:50:59; Latitude=22.2318; Longitude=156.855; Temperature=23.0028; Salinity=35.1175; Particulate_carbon=2.35172 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S64C1-DCM_A.mzML 190715_Smp_FK180310S64C1-DCM_A.mzML Sample_type:Sample | Station:64 | Depth:DCM | Absolute_depth:115 | Sea_level_anomaly:-12.1225 Time_sampled=2018-04-04 10:50:59; Latitude=22.1359; Longitude=156.984; Temperature=21.1728; Salinity=35.1679; Particulate_carbon=1.7286 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S64C1-DCM_B.mzML 190715_Smp_FK180310S64C1-DCM_B.mzML Sample_type:Sample | Station:64 | Depth:DCM | Absolute_depth:115 | Sea_level_anomaly:-12.1225 Time_sampled=2018-04-04 10:50:59; Latitude=22.1359; Longitude=156.984; Temperature=21.1728; Salinity=35.1679; Particulate_carbon=1.7286 SUBJECT_SAMPLE_FACTORS 190715_Smp_FK180310S64C1-DCM_C.mzML 190715_Smp_FK180310S64C1-DCM_C.mzML Sample_type:Sample | Station:64 | Depth:DCM | Absolute_depth:115 | Sea_level_anomaly:-12.1225 Time_sampled=2018-04-04 10:50:59; Latitude=22.1359; Longitude=156.984; Temperature=21.1728; Salinity=35.1679; Particulate_carbon=1.7286 SUBJECT_SAMPLE_FACTORS 190715_Blk_KM1906U14-Blk_C.mzML 190715_Blk_KM1906U14-Blk_C.mzML Sample_type:Blank | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA SUBJECT_SAMPLE_FACTORS 190715_Poo_TruePooFK180310_Full1.mzML 190715_Poo_TruePooFK180310_Full1.mzML Sample_type:QC Pooled | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA SUBJECT_SAMPLE_FACTORS 190715_Poo_TruePooFK180310_Full2.mzML 190715_Poo_TruePooFK180310_Full2.mzML Sample_type:QC Pooled | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA SUBJECT_SAMPLE_FACTORS 190715_Poo_TruePooFK180310_Full3.mzML 190715_Poo_TruePooFK180310_Full3.mzML Sample_type:QC Pooled | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA SUBJECT_SAMPLE_FACTORS 190715_Poo_TruePooFK180310_Half1.mzML 190715_Poo_TruePooFK180310_Half1.mzML Sample_type:QC Pooled | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA SUBJECT_SAMPLE_FACTORS 190715_Poo_TruePooFK180310_Half2.mzML 190715_Poo_TruePooFK180310_Half2.mzML Sample_type:QC Pooled | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA SUBJECT_SAMPLE_FACTORS 190715_Poo_TruePooFK180310_Half3.mzML 190715_Poo_TruePooFK180310_Half3.mzML Sample_type:QC Pooled | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA SUBJECT_SAMPLE_FACTORS 190715_Std_4uMStdsMix1InH2O_1.mzML 190715_Std_4uMStdsMix1InH2O_1.mzML Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA SUBJECT_SAMPLE_FACTORS 190715_Std_4uMStdsMix1InH2O_2.mzML 190715_Std_4uMStdsMix1InH2O_2.mzML Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA SUBJECT_SAMPLE_FACTORS 190715_Std_4uMStdsMix1InMatrix_1.mzML 190715_Std_4uMStdsMix1InMatrix_1.mzML Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA SUBJECT_SAMPLE_FACTORS 190715_Std_4uMStdsMix1InMatrix_2.mzML 190715_Std_4uMStdsMix1InMatrix_2.mzML Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA SUBJECT_SAMPLE_FACTORS 190715_Std_4uMStdsMix2InH2O_1.mzML 190715_Std_4uMStdsMix2InH2O_1.mzML Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA SUBJECT_SAMPLE_FACTORS 190715_Std_4uMStdsMix2InH2O_2.mzML 190715_Std_4uMStdsMix2InH2O_2.mzML Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA SUBJECT_SAMPLE_FACTORS 190715_Std_4uMStdsMix2InMatrix_1.mzML 190715_Std_4uMStdsMix2InMatrix_1.mzML Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA SUBJECT_SAMPLE_FACTORS 190715_Std_4uMStdsMix2InMatrix_2.mzML 190715_Std_4uMStdsMix2InMatrix_2.mzML Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA SUBJECT_SAMPLE_FACTORS 190715_Std_H2OinMatrix_1.mzML 190715_Std_H2OinMatrix_1.mzML Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA SUBJECT_SAMPLE_FACTORS 190715_Std_H2OinMatrix_2.mzML 190715_Std_H2OinMatrix_2.mzML Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Time_sampled=NA; Latitude=NA; Longitude=NA; Temperature=NA; Salinity=NA; Particulate_carbon=NA #COLLECTION CO:COLLECTION_SUMMARY Samples were collected aboard the R/V Falkor in March and April 2018. Water was CO:COLLECTION_SUMMARY collected with Niskin bottles attached to the CTD from the surface and the deep CO:COLLECTION_SUMMARY chlorophyll maximum (DCM) in both the morning (~6AM) and evening (~6PM). Water CO:COLLECTION_SUMMARY was filtered onto a 0.2um pore size filter under gentle vacuum pressure prior to CO:COLLECTION_SUMMARY flash-freezing in LN2 before storage at -80 in precombusted aluminum foil. CO:COLLECTION_SUMMARY Samples were collected from two locations, one in a cyclonic eddy (-13cm, CO:COLLECTION_SUMMARY stations 62 and 64) and one in an anticyclonic eddy (+20cm, stations 77 and 80) CO:COLLECTION_SUMMARY three days apart. CO:SAMPLE_TYPE Suspended Marine Particulate Matter CO:COLLECTION_METHOD CTD Niskin Bottle CO:COLLECTION_LOCATION North Pacific CO:VOLUMEORAMOUNT_COLLECTED 10L CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY No experimental manipulations were performed on the samples. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Polar metabolites were extracted using a modified Bligh−Dyer extraction using SP:SAMPLEPREP_SUMMARY 1:1 methanol/water and dichloromethane. A methodological blank was extracted and SP:SAMPLEPREP_SUMMARY analyzed alongside. Isotope-labeled internal standards were added both before SP:SAMPLEPREP_SUMMARY and after the extraction for all samples, blanks, and pooled samples. To SP:SAMPLEPREP_SUMMARY evaluate the effect of obscuring variation due to different matrix strengths and SP:SAMPLEPREP_SUMMARY analytical drift, pooled samples were run at both full and half concentration SP:SAMPLEPREP_SUMMARY (diluted with water) three times during the run. SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACTION_METHOD Bligh-Dyer SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY A SeQuant ZIC-pHILIC column (5 um particle size, 2.1 mm x 150 mm, from CH:CHROMATOGRAPHY_SUMMARY Millipore) was used with 10 mM ammonium carbonate in 85:15 acetonitrile to water CH:CHROMATOGRAPHY_SUMMARY (Solvent A) and 10 mM ammonium carbonate in 85:15 water to acetonitrile (Solvent CH:CHROMATOGRAPHY_SUMMARY B) at a flow rate of 0.15 mL/min. This column was compared with a Waters UPLC CH:CHROMATOGRAPHY_SUMMARY BEH amide and a Millipore cHILIC column; the pHILIC showed superior CH:CHROMATOGRAPHY_SUMMARY reproducibility and peak shapes. The column was held at 100% A for 2 minutes, CH:CHROMATOGRAPHY_SUMMARY ramped to 64% B over 18 minutes, ramped to 100% B over 1 minute, held at 100% B CH:CHROMATOGRAPHY_SUMMARY for 5 minutes, and equilibrated at 100% A for 25 minutes (50 minutes total). The CH:CHROMATOGRAPHY_SUMMARY column was maintained at 30 C. The injection volume was 2 µL for samples and CH:CHROMATOGRAPHY_SUMMARY standard mixes. When starting a batch, the column was equilibrated at the CH:CHROMATOGRAPHY_SUMMARY starting conditions for at least 30 minutes. To improve the performance of the CH:CHROMATOGRAPHY_SUMMARY HILIC column, we maintained the same injection volume, kept the instrument CH:CHROMATOGRAPHY_SUMMARY running water blanks between samples as necessary, and injected standards in a CH:CHROMATOGRAPHY_SUMMARY representative matrix in addition to standards in water. After each batch, the CH:CHROMATOGRAPHY_SUMMARY column was flushed with 10 mM ammonium carbonate in 85:15 water to acetonitrile CH:CHROMATOGRAPHY_SUMMARY for 20 to 30 minutes. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Waters Acquity I-Class CH:COLUMN_NAME SeQuant ZIC-HILIC (150 x 2.1mm, 5um) CH:SOLVENT_A 85% acetonitrile/15% water CH:SOLVENT_B 15% acetonitrile/85% water CH:FLOW_GRADIENT 100% A for 2 minutes, ramped to 64% B over 18 minutes, ramped to 100% B over 1 CH:FLOW_GRADIENT minute, held at 100% B for 5 minutes, and equilibrated at 100% A for 25 minutes CH:FLOW_GRADIENT (50 minutes total) CH:FLOW_RATE 0.15 mL/min CH:COLUMN_TEMPERATURE 30 #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 Metabolomic data was collected on a Thermo Q Exactive HF hybrid Orbitrap (QE) MS:MS_COMMENTS mass spectrometer. The capillary and auxiliary gas heater temperatures were MS:MS_COMMENTS maintained at 320°C and 100°C, respectively. The S-lens RF level was kept at MS:MS_COMMENTS 65, the H-ESI voltage was set to 3.3 kV and sheath gas, auxiliary gas, and sweep MS:MS_COMMENTS gas flow rates were set at 16, 3, and 1, respectively. Polarity switching was MS:MS_COMMENTS used with a scan range of 60 to 900 m/z and a resolution of 60,000. Calibration MS:MS_COMMENTS was performed every 3-4 days at a target mass of 200 m/z. All files were then MS:MS_COMMENTS converted to an open-source mzML format and centroided via Proteowizard’s MS:MS_COMMENTS msConvert tool. Skyline was used to for manual integration and QC of quantified MS:MS_COMMENTS data, while XCMS was used with a custom QC method for peak area data with the MS:MS_COMMENTS model trained on positive mode data applied to these negative mode results. MS:MS_RESULTS_FILE ST002788_AN004537_Results.txt UNITS:normalized peak area per liter of seawater filtered Has m/z:Yes Has RT:Yes RT units:Minutes #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nM MS_METABOLITE_DATA_START Samples 190715_Std_4uMStdsMix1InH2O_1.mzML 190715_Std_4uMStdsMix1InH2O_2.mzML 190715_Std_4uMStdsMix1InMatrix_1.mzML 190715_Std_4uMStdsMix1InMatrix_2.mzML 190715_Std_4uMStdsMix2InH2O_1.mzML 190715_Std_4uMStdsMix2InH2O_2.mzML 190715_Std_4uMStdsMix2InMatrix_1.mzML 190715_Std_4uMStdsMix2InMatrix_2.mzML 190715_Std_H2OinMatrix_2.mzML 190715_Std_H2OinMatrix_1.mzML 190715_Blk_KM1906U14-Blk_C.mzML 190715_Poo_TruePooFK180310_Full1.mzML 190715_Poo_TruePooFK180310_Full2.mzML 190715_Poo_TruePooFK180310_Full3.mzML 190715_Poo_TruePooFK180310_Half1.mzML 190715_Poo_TruePooFK180310_Half2.mzML 190715_Poo_TruePooFK180310_Half3.mzML 190715_Smp_FK180310S62C1-25m_A.mzML 190715_Smp_FK180310S62C1-25m_B.mzML 190715_Smp_FK180310S62C1-25m_C.mzML 190715_Smp_FK180310S62C1-DCM_A.mzML 190715_Smp_FK180310S62C1-DCM_B.mzML 190715_Smp_FK180310S62C1-DCM_C.mzML 190715_Smp_FK180310S64C1-25m_A.mzML 190715_Smp_FK180310S64C1-25m_B.mzML 190715_Smp_FK180310S64C1-25m_C.mzML 190715_Smp_FK180310S64C1-DCM_A.mzML 190715_Smp_FK180310S64C1-DCM_B.mzML 190715_Smp_FK180310S64C1-DCM_C.mzML 190715_Smp_FK180310S77C1-25m_A.mzML 190715_Smp_FK180310S77C1-25m_B.mzML 190715_Smp_FK180310S77C1-25m_C.mzML 190715_Smp_FK180310S77C1-DCM_A.mzML 190715_Smp_FK180310S77C1-DCM_B.mzML 190715_Smp_FK180310S77C1-DCM_C.mzML 190715_Smp_FK180310S80C1-25m_A.mzML 190715_Smp_FK180310S80C1-25m_B.mzML 190715_Smp_FK180310S80C1-25m_C.mzML 190715_Smp_FK180310S80C1-DCM_A.mzML 190715_Smp_FK180310S80C1-DCM_B.mzML 190715_Smp_FK180310S80C1-DCM_C.mzML Factors Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:Standard mix | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:Blank | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:QC Pooled | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:QC Pooled | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:QC Pooled | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:QC Pooled | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:QC Pooled | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:QC Pooled | Station:NA | Depth:NA | Absolute_depth:NA | Sea_level_anomaly:NA Sample_type:Sample | Station:62 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:-12.48 Sample_type:Sample | Station:62 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:-12.48 Sample_type:Sample | Station:62 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:-12.48 Sample_type:Sample | Station:62 | Depth:DCM | Absolute_depth:98 | Sea_level_anomaly:-13.6 Sample_type:Sample | Station:62 | Depth:DCM | Absolute_depth:98 | Sea_level_anomaly:-13.6 Sample_type:Sample | Station:62 | Depth:DCM | Absolute_depth:98 | Sea_level_anomaly:-13.6 Sample_type:Sample | Station:64 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:-12.48 Sample_type:Sample | Station:64 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:-12.48 Sample_type:Sample | Station:64 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:-12.48 Sample_type:Sample | Station:64 | Depth:DCM | Absolute_depth:115 | Sea_level_anomaly:-12.1225 Sample_type:Sample | Station:64 | Depth:DCM | Absolute_depth:115 | Sea_level_anomaly:-12.1225 Sample_type:Sample | Station:64 | Depth:DCM | Absolute_depth:115 | Sea_level_anomaly:-12.1225 Sample_type:Sample | Station:77 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:20.8044 Sample_type:Sample | Station:77 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:20.8044 Sample_type:Sample | Station:77 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:20.8044 Sample_type:Sample | Station:77 | Depth:DCM | Absolute_depth:125 | Sea_level_anomaly:19.87 Sample_type:Sample | Station:77 | Depth:DCM | Absolute_depth:125 | Sea_level_anomaly:19.87 Sample_type:Sample | Station:77 | Depth:DCM | Absolute_depth:125 | Sea_level_anomaly:19.87 Sample_type:Sample | Station:80 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:20.8044 Sample_type:Sample | Station:80 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:20.8044 Sample_type:Sample | Station:80 | Depth:Surface | Absolute_depth:25 | Sea_level_anomaly:20.8044 Sample_type:Sample | Station:80 | Depth:DCM | Absolute_depth:120 | Sea_level_anomaly:21.03 Sample_type:Sample | Station:80 | Depth:DCM | Absolute_depth:120 | Sea_level_anomaly:21.03 Sample_type:Sample | Station:80 | Depth:DCM | Absolute_depth:120 | Sea_level_anomaly:21.03 (2R)-3-sulfopropanediol NA NA 0.07623 0.06059 1.021 1.044 0.4037 0.3864 0.06674 0.08343 NA 0.0575 0.05806 0.05844 0.05493 0.03943 0.04726 0.1094 0.04871 0.06538 0.1132 0.5176 0.1212 0.0424 0.0759 0.05156 0.09176 0.1126 0.1167 0.02705 0.02764 0.01491 0.1526 0.08737 0.05025 0.02736 0.07617 0.05254 0.03782 0.08405 0.1122 Pyroglutamic acid 0.3573 0.3326 0.5999 0.5241 2.127e-05 NA 0.2372 0.2125 0.2072 0.2768 0.05881 0.1624 0.1249 0.1372 0.1536 0.1379 0.1125 0.1474 0.09714 0.308 0.08918 0.1992 0.1919 0.1227 0.1258 0.1714 0.06559 0.1266 0.1122 0.1345 0.1097 0.259 0.1294 0.04499 0.153 0.1677 0.1293 0.1528 0.05499 0.1052 0.1719 AMP NA NA 0.001566 0.00237 NA NA 0.5098 0.999 0.002022 NA NA 0.03154 0.02532 0.0348 0.02138 0.02489 0.03594 0.06451 0.02268 0.178 0 0.04288 0.01329 0.01969 0.03075 0.2031 0.003622 0.01836 0.01406 0.006448 0 0.006666 0.0005242 0.003922 0.007322 0.002006 0.02362 0.0479 0.001828 0.009006 0.01527 2-Hydroxyethanesulfonate NA NA 0.1262 0.1397 NA NA 0.4871 0.5517 0.1471 0.1399 0.005158 0.05967 0.0517 0.05536 0.05402 0.04879 0.05438 0.0742 0.04614 0.2424 0.03543 0.09519 0.05668 0.07249 0.05386 0.1173 0.0298 0.04616 0.05112 0.04049 0.03997 0.02171 0.02854 0.02014 0.02829 0.03334 0.05085 0.06381 0.01676 0.03756 0.04465 Malic acid 0.3973 0.2848 0.467 0.3248 0.02386 0.006001 0.09729 0.05982 0.06019 0.09163 0.01967 0.05342 0.05203 0.06395 0.06062 0.08843 0.06077 0.07537 0.07326 0.08236 0.05073 0.1045 0.07023 0.06924 0.07189 0.06446 0.05177 0.05414 0.07008 0.0567 0.04606 0.05148 0.05732 0.05353 0.06813 0.06576 0.08195 0.07215 0.03888 0.07578 0.06178 Sucrose NA NA 0.5824 0.725 NA NA 0.1501 0.1387 0.1374 0.1466 0.03394 0.1789 0.1628 0.1738 0.1671 0.1718 0.166 0.1839 0.1357 0.1661 0.02714 0.03843 0.04884 0.318 0.287 0.321 0.1355 0.1584 0.1314 0.1177 0.1174 0.1909 0.0509 0.02666 0.0319 0.211 0.2494 0.2547 0.1457 0.3612 NA Taurine NA NA 0.09289 0.09332 NA NA 0.3923 0.4295 0.08632 0.08816 0.006899 0.102 0.09377 0.1005 0.1167 0.1107 0.1041 0.1241 0.06072 0.4825 0.05064 0.2302 0.08265 0.09987 0.1325 0.2123 0.0443 0.09777 0.08364 0.05702 0.07094 0.0406 0.05355 0.06292 0.05192 0.04944 0.07572 0.2227 0.03269 0.06618 0.07566 Trehalose 0.0006444 0 0.004686 0.002619 0.3411 0.2368 0.3692 0.2819 0.002212 0.008819 0.001892 0.004014 0.006906 0.006039 0.001572 0.001065 0 0.001233 0 0.007317 0 0.00778 0.0009122 0.01587 0.01598 0.01997 0.003898 0.006521 0.005651 0.006817 0.001312 0.0005974 0.0007464 0 0.0006634 0.00594 0.01515 0.009641 0.001671 0.007191 0.01282 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name formula stan_mz stan_rt_expected which_standard_mix concentration_added kegg_id (2R)-3-sulfopropanediol C3H8O5S 155.001422 10.67 Mix2 4 cpd:C19675 Pyroglutamic acid C5H7NO3 128.034768 7.97 Mix1 4 cpd:C01879 AMP C10H14N5O7P 346.055262 11.585 Mix2 4 cpd:C00020 2-Hydroxyethanesulfonate C2H6O4S 124.990857 7.2 Mix2 4 cpd:C05123 Malic acid C4H6O5 133.0137 11.97 Mix1 4 cpd:C00149 Sucrose C12H22O11 341.10839 11.93 Mix1 5 cpd:C00089 Taurine C2H7NO3S 124.006841 11.13 Mix2 4 cpd:C00245 Trehalose C12H22O11 341.10839 13 Mix2 4 cpd:C01083 METABOLITES_END #END