#METABOLOMICS WORKBENCH TruxalCarlson_20200324_121313 DATATRACK_ID:1951 STUDY_ID:ST001393 ANALYSIS_ID:AN002329 PROJECT_ID:PR000956 VERSION 1 CREATED_ON June 4, 2020, 4:06 pm #PROJECT PR:PROJECT_TITLE Sea-ice diatom compatible solute shifts PR:PROJECT_TYPE Marine Metabolomics PR:PROJECT_SUMMARY Sea-ice algae provide an important source of primary production in polar PR:PROJECT_SUMMARY regions, yet we have limited understanding of their responses to the seasonal PR:PROJECT_SUMMARY cycling of temperature and salinity. Using a targeted liquid chromatography-mass PR:PROJECT_SUMMARY spectrometry-based metabolomics approach, we found that axenic cultures of the PR:PROJECT_SUMMARY Antarctic sea-ice diatom, Nitzschia lecointei, displayed large differences in PR:PROJECT_SUMMARY their metabolomes when grown in a matrix of conditions that included PR:PROJECT_SUMMARY temperatures of –1 and 4°C, and salinities of 32 and 41, despite relatively PR:PROJECT_SUMMARY small changes in growth rate. Temperature exerted a greater effect than salinity PR:PROJECT_SUMMARY on cellular metabolite pool sizes, though the N- or S-containing compatible PR:PROJECT_SUMMARY solutes, 2,3-dihydroxypropane-1-sulfonate (DHPS), glycine betaine (GBT), PR:PROJECT_SUMMARY dimethylsulfoniopropionate (DMSP), and proline responded strongly to both PR:PROJECT_SUMMARY temperature and salinity, suggesting complexity in their control. We saw the PR:PROJECT_SUMMARY largest (> 4 fold) response to salinity for proline. DHPS, a rarely studied but PR:PROJECT_SUMMARY potential compatible solute, reached the highest intracellular compatible solute PR:PROJECT_SUMMARY concentrations of ~ 85 mM. When comparing the culture findings to natural Arctic PR:PROJECT_SUMMARY sea-ice diatom communities, we found extensive overlap in metabolite profiles, PR:PROJECT_SUMMARY highlighting the relevance of culture-based studies to probe environmental PR:PROJECT_SUMMARY questions. Large changes in sea-ice diatom metabolomes and compatible solutes PR:PROJECT_SUMMARY over a seasonal cycle could be significant components of biogeochemical cycling PR:PROJECT_SUMMARY within sea ice. PR:INSTITUTE University of Washington PR:DEPARTMENT School of Oceanography PR:LABORATORY Ingalls Lab PR:LAST_NAME Dawson PR:FIRST_NAME Hannah PR:ADDRESS 1501 NE Boat Street, Marine Science Building, Room G, Seattle, WA 98195 PR:EMAIL hmdawson@uw.edu PR:PHONE 2062216750 PR:FUNDING_SOURCE Booth Foundation, NSF, UW Graduate Top Scholar Award, Gordon and Betty Moore PR:FUNDING_SOURCE Foundation PR:PUBLICATIONS Dawson et al., Elementa #STUDY ST:STUDY_TITLE Sea-ice diatom compatible solute shifts ST:STUDY_TYPE Compatible solutes were quantified in sea-ice diatoms ST:STUDY_SUMMARY Sea-ice algae provide an important source of primary production in polar ST:STUDY_SUMMARY regions, yet we have limited understanding of their responses to the seasonal ST:STUDY_SUMMARY cycling of temperature and salinity. Using a targeted liquid chromatography-mass ST:STUDY_SUMMARY spectrometry-based metabolomics approach, we found that axenic cultures of the ST:STUDY_SUMMARY Antarctic sea-ice diatom, Nitzschia lecointei, displayed large differences in ST:STUDY_SUMMARY their metabolomes when grown in a matrix of conditions that included ST:STUDY_SUMMARY temperatures of –1 and 4°C, and salinities of 32 and 41, despite relatively ST:STUDY_SUMMARY small changes in growth rate. Temperature exerted a greater effect than salinity ST:STUDY_SUMMARY on cellular metabolite pool sizes, though the N- or S-containing compatible ST:STUDY_SUMMARY solutes, 2,3-dihydroxypropane-1-sulfonate (DHPS), glycine betaine (GBT), ST:STUDY_SUMMARY dimethylsulfoniopropionate (DMSP), and proline responded strongly to both ST:STUDY_SUMMARY temperature and salinity, suggesting complexity in their control. We saw the ST:STUDY_SUMMARY largest (> 4 fold) response to salinity for proline. DHPS, a rarely studied but ST:STUDY_SUMMARY potential compatible solute, reached the highest intracellular compatible solute ST:STUDY_SUMMARY concentrations of ~ 85 mM. When comparing the culture findings to natural Arctic ST:STUDY_SUMMARY sea-ice diatom communities, we found extensive overlap in metabolite profiles, ST:STUDY_SUMMARY highlighting the relevance of culture-based studies to probe environmental ST:STUDY_SUMMARY questions. Large changes in sea-ice diatom metabolomes and compatible solutes ST:STUDY_SUMMARY over a seasonal cycle could be significant components of biogeochemical cycling ST:STUDY_SUMMARY within sea ice. ST:INSTITUTE University of Washington ST:DEPARTMENT School of Oceanography ST:LABORATORY Ingalls Lab ST:LAST_NAME Dawson ST:FIRST_NAME Hannah ST:ADDRESS 1501 NE Boat Street, Marine Science Building, Room G, Seattle, WA 98195 ST:EMAIL hmdawson@uw.edu ST:PHONE 2062216750 ST:PUBLICATIONS Dawson et al., Elementa #SUBJECT SU:SUBJECT_TYPE Other SU:SUBJECT_SPECIES Nitzschia lecointei SU:TAXONOMY_ID 186028 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 - 32ppt-1C_A Type:Smp | Salinity:32 | Temp_degC:-1 Replicate=A; RFU=605.6; Vol_L=0.07; RAW_FILE_NAME=170410_Smp_32ppt-1C_A;170413_Smp_40ppt4C_C;170410_Smp_32ppt-1C_A SUBJECT_SAMPLE_FACTORS - 32ppt-1C_B Type:Smp | Salinity:32 | Temp_degC:-1 Replicate=B; RFU=551.2; Vol_L=0.07; RAW_FILE_NAME=170410_Smp_32ppt-1C_B;170413_Smp_32ppt-1C_B;170410_Smp_32ppt-1C_B SUBJECT_SAMPLE_FACTORS - 32ppt-1C_C Type:Smp | Salinity:32 | Temp_degC:-1 Replicate=C; RFU=550.6; Vol_L=0.07; RAW_FILE_NAME=170410_Smp_32ppt-1C_C;170413_Smp_32ppt-1C_C;170410_Smp_32ppt-1C_C SUBJECT_SAMPLE_FACTORS - 32ppt4C_A Type:Smp | Salinity:32 | Temp_degC:4 Replicate=A; RFU=847.1; Vol_L=0.07; RAW_FILE_NAME=170410_Smp_32ppt4C_A;170413_Smp_32ppt4C_B;170410_Smp_32ppt4C_A SUBJECT_SAMPLE_FACTORS - 32ppt4C_B Type:Smp | Salinity:32 | Temp_degC:4 Replicate=B; RFU=967.1; Vol_L=0.07; RAW_FILE_NAME=170410_Smp_32ppt4C_B;170413_Smp_32ppt4C_A;170410_Smp_32ppt4C_B SUBJECT_SAMPLE_FACTORS - 32ppt4C_C Type:Smp | Salinity:32 | Temp_degC:4 Replicate=C; RFU=918.5; Vol_L=0.07; RAW_FILE_NAME=170410_Smp_32ppt4C_C;170413_Smp_32ppt4C_C;170410_Smp_32ppt4C_C SUBJECT_SAMPLE_FACTORS - 40ppt-1C_A Type:Smp | Salinity:40 | Temp_degC:-1 Replicate=A; RFU=860.2; Vol_L=0.07; RAW_FILE_NAME=170410_Smp_40ppt-1C_A;170413_Smp_40ppt-1C_A;170410_Smp_40ppt-1C_A SUBJECT_SAMPLE_FACTORS - 40ppt-1C_B Type:Smp | Salinity:40 | Temp_degC:-1 Replicate=B; RFU=681.6; Vol_L=0.07; RAW_FILE_NAME=170410_Smp_40ppt-1C_B;170413_Smp_40ppt4C_B;170410_Smp_40ppt-1C_B SUBJECT_SAMPLE_FACTORS - 40ppt-1C_C Type:Smp | Salinity:40 | Temp_degC:-1 Replicate=C; RFU=814.3; Vol_L=0.07; RAW_FILE_NAME=170410_Smp_40ppt-1C_C;170413_Smp_40ppt-1C_C;170410_Smp_40ppt-1C_C SUBJECT_SAMPLE_FACTORS - 40ppt4C_A Type:Smp | Salinity:40 | Temp_degC:4 Replicate=A; RFU=581.8; Vol_L=0.07; RAW_FILE_NAME=170410_Smp_40ppt4C_A;170413_Smp_40ppt4C_A;170410_Smp_40ppt4C_A SUBJECT_SAMPLE_FACTORS - 40ppt4C_B Type:Smp | Salinity:40 | Temp_degC:4 Replicate=B; RFU=681.6; Vol_L=0.07; RAW_FILE_NAME=170410_Smp_40ppt4C_B;170413_Smp_40ppt-1C_B;170410_Smp_40ppt4C_B SUBJECT_SAMPLE_FACTORS - 40ppt4C_C Type:Smp | Salinity:40 | Temp_degC:4 Replicate=C; RFU=662; Vol_L=0.07; RAW_FILE_NAME=170410_Smp_40ppt4C_C;170413_Smp_32ppt-1C_A;170410_Smp_40ppt4C_C SUBJECT_SAMPLE_FACTORS - ASWFilterBlk_1 Type:Blk | Salinity:NA | Temp_degC:NA Replicate=1; RFU=NA; Vol_L=0.3; RAW_FILE_NAME=170612_Blk_ASWFilterBlk_1;170615_Blk_ASWFilterBlk_1;170612_Blk_ASWFilterBlk_1 SUBJECT_SAMPLE_FACTORS - ASWFilterBlk_2 Type:Blk | Salinity:NA | Temp_degC:NA Replicate=2; RFU=NA; Vol_L=0.3; RAW_FILE_NAME=170612_Blk_ASWFilterBlk_2;170615_Blk_ASWFilterBlk_2;170612_Blk_ASWFilterBlk_2 SUBJECT_SAMPLE_FACTORS - ASWFilterBlk_3 Type:Blk | Salinity:NA | Temp_degC:NA Replicate=3; RFU=NA; Vol_L=0.3; RAW_FILE_NAME=170612_Blk_ASWFilterBlk_3;170615_Blk_ASWFilterBlk_3;170612_Blk_ASWFilterBlk_3 SUBJECT_SAMPLE_FACTORS - MediaBlk_ppt32 Type:Blk | Salinity:32 | Temp_degC:NA Replicate=ppt32; RFU=1; Vol_L=0.07; RAW_FILE_NAME=170410_Blk_MediaBlk_ppt32;170413_Blk_MediaBlk_ppt32;170410_Blk_MediaBlk_ppt32 SUBJECT_SAMPLE_FACTORS - MediaBlk_ppt40 Type:Blk | Salinity:40 | Temp_degC:NA Replicate=ppt40; RFU=1; Vol_L=0.07; RAW_FILE_NAME=170410_Blk_MediaBlk_ppt40;170413_Blk_MediaBlk_ppt40;170410_Blk_MediaBlk_ppt40 SUBJECT_SAMPLE_FACTORS - S2C_4 Type:Smp | Salinity:NA | Temp_degC:NA Replicate=4; RFU=NA; Vol_L=0.1671; RAW_FILE_NAME=170612_Smp_S2C_4;170615_Smp_S2C_4;170612_Smp_S2C_4 SUBJECT_SAMPLE_FACTORS - S2C_5 Type:Smp | Salinity:NA | Temp_degC:NA Replicate=5; RFU=NA; Vol_L=0.2486; RAW_FILE_NAME=170612_Smp_S2C_5;170615_Smp_S2C_5;170612_Smp_S2C_5 SUBJECT_SAMPLE_FACTORS - S2C_6 Type:Smp | Salinity:NA | Temp_degC:NA Replicate=6; RFU=NA; Vol_L=0.2049; RAW_FILE_NAME=170612_Smp_S2C_6;170615_Smp_S2C_6;170612_Smp_S2C_6 #COLLECTION CO:COLLECTION_SUMMARY Cultured diatom cells at different salinities and temperatures grown to CO:COLLECTION_SUMMARY exponential phase were filtered onto 0.2-micron filters and extracted for CO:COLLECTION_SUMMARY metabolites as described in methods. Three dedicated ice cores were sampled from CO:COLLECTION_SUMMARY the Chukchi Sea near Utqiaġvik, AK. The bottom 5-cm sections were placed in CO:COLLECTION_SUMMARY polycarbonate tubs, allowed to melt at 4°C in artificial seawater, and filtered CO:COLLECTION_SUMMARY onto 0.2-micron filters. Filters were extracted for metabolites as described in CO:COLLECTION_SUMMARY methods. All filters were frozen in liquid nitrogen immediately after filtration CO:COLLECTION_SUMMARY and stored in a -80 C freezer until extraction. CO:SAMPLE_TYPE Diatom cells/Particulate matter from sea ice cores CO:STORAGE_CONDITIONS Described in summary #TREATMENT TR:TREATMENT_SUMMARY Diatom cells were cultured in a matrix of two temperatures (–1°C and 4°C) TR:TREATMENT_SUMMARY and two salinities (32 and 40) in triplicate. There was no treatment for the sea TR:TREATMENT_SUMMARY ice cores – this was a study of how the cultured diatoms compare to the TR:TREATMENT_SUMMARY diatom-dominated Arctic sea-ice communities. #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 protocol, data from culture samples #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Normalized Peak Area Per RFU MS_METABOLITE_DATA_START Samples 32ppt-1C_A 32ppt-1C_B 32ppt-1C_C 32ppt4C_A 32ppt4C_B 32ppt4C_C 40ppt-1C_A 40ppt-1C_B 40ppt-1C_C 40ppt4C_A 40ppt4C_B 40ppt4C_C MediaBlk_ppt32 MediaBlk_ppt40 Factors Type:Smp | Salinity:32 | Temp_degC:-1 Type:Smp | Salinity:32 | Temp_degC:-1 Type:Smp | Salinity:32 | Temp_degC:-1 Type:Smp | Salinity:32 | Temp_degC:4 Type:Smp | Salinity:32 | Temp_degC:4 Type:Smp | Salinity:32 | Temp_degC:4 Type:Smp | Salinity:40 | Temp_degC:-1 Type:Smp | Salinity:40 | Temp_degC:-1 Type:Smp | Salinity:40 | Temp_degC:-1 Type:Smp | Salinity:40 | Temp_degC:4 Type:Smp | Salinity:40 | Temp_degC:4 Type:Smp | Salinity:40 | Temp_degC:4 Type:Blk | Salinity:32 | Temp_degC:NA Type:Blk | Salinity:40 | Temp_degC:NA DHPS 25874543.94 25489446.27 26512505.63 14887867.22 12509887.49 15735596.73 20589709.18 28831546.29 24234743.79 24751035.71 21576599.44 20423500.57 14220 13070 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name quantitated m/z CHEBI KEGGNAME MS_method KEGG ID DHPS 155.001422 CHEBI:60997 (R)-2,3-Dihydroxypropane-1-sulfonate HILIC_QE_Neg C19675 METABOLITES_END #END