#METABOLOMICS WORKBENCH uchimiya_20221017_095808 DATATRACK_ID:3514 STUDY_ID:ST002321 ANALYSIS_ID:AN003788 PROJECT_ID:PR001487 VERSION 1 CREATED_ON October 17, 2022, 11:30 am #PROJECT PR:PROJECT_TITLE 13C NMR metabolomics: integrating J-resolved STOCSY and INADEQUATE PR:PROJECT_SUMMARY Robust annotation of metabolites remains a challenging task in metabolomics. PR:PROJECT_SUMMARY This study introduces an approach that uses 13C homonuclear J-resolved PR:PROJECT_SUMMARY experiment (JRES), statistical total correlation spectroscopy (STOCSY), and 2D PR:PROJECT_SUMMARY incredible natural abundance double-quantum experiment (INADEQUATE) PR:PROJECT_SUMMARY complementarily, to obtain robust molecular structure information based on 13C PR:PROJECT_SUMMARY NMR with less experiment time. This approach was tested using the endometabolome PR:PROJECT_SUMMARY from a model marine phytoplankton strain, varying the settings of incubation PR:PROJECT_SUMMARY temperature, nutrient condition, and the presence of co-culturing bacteria. PR:INSTITUTE University of Georgia PR:LAST_NAME Uchimiya PR:FIRST_NAME Mario PR:ADDRESS 315 Riverbend Rd, Athens, GA, 30602, USA PR:EMAIL mario.uchimiya@uga.edu PR:PHONE (706) 542-8387 PR:FUNDING_SOURCE NSF (grant numbers 1948104, OCE-2019589, and 1946970), NIH (5R01GM120151-04) PR:CONTRIBUTORS Edison Lab, Moran Lab #STUDY ST:STUDY_TITLE 13C NMR metabolomics: integrating J-resolved STOCSY and INADEQUATE ST:STUDY_SUMMARY Robust annotation of metabolites remains a challenging task in metabolomics. ST:STUDY_SUMMARY This study introduces an approach that uses 13C homonuclear J-resolved ST:STUDY_SUMMARY experiment (JRES), statistical total correlation spectroscopy (STOCSY), and 2D ST:STUDY_SUMMARY incredible natural abundance double-quantum experiment (INADEQUATE) ST:STUDY_SUMMARY complementarily, to obtain robust molecular structure information based on 13C ST:STUDY_SUMMARY NMR with less experiment time. This approach was tested using the endometabolome ST:STUDY_SUMMARY from a model marine phytoplankton strain, varying the settings of incubation ST:STUDY_SUMMARY temperature, nutrient condition, and the presence of co-culturing bacteria. ST:INSTITUTE University of Georgia ST:LAST_NAME Uchimiya ST:FIRST_NAME Mario ST:ADDRESS 315 Riverbend Rd, Athens, GA, 30602, USA ST:EMAIL mario.uchimiya@uga.edu ST:PHONE (706) 542-8387 #SUBJECT SU:SUBJECT_TYPE Other organism SU:SUBJECT_SPECIES Thalassiosira pseudonana SU:TAXONOMY_ID 296543 #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 - 6 Factor_1 (temperature):14.0 | Factor_2 (bacteria presence):1.0 RAW_FILE_NAME=6; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=14°C Co-culture; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 9 Factor_1 (temperature):14.0 | Factor_2 (bacteria presence):1.0 RAW_FILE_NAME=9; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=14°C Co-culture; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 12 Factor_1 (temperature):14.0 | Factor_2 (bacteria presence):1.0 RAW_FILE_NAME=12; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=14°C Co-culture; Biological replicate=3 SUBJECT_SAMPLE_FACTORS - 15 Factor_1 (temperature):14.0 | Factor_2 (bacteria presence):1.0 RAW_FILE_NAME=15; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=14°C Co-culture; Biological replicate=4 SUBJECT_SAMPLE_FACTORS - 18 Factor_1 (temperature):20.0 | Factor_2 (bacteria presence):1.0 RAW_FILE_NAME=18; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=20°C Co-culture; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 21 Factor_1 (temperature):20.0 | Factor_2 (bacteria presence):1.0 RAW_FILE_NAME=21; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=20°C Co-culture; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 24 Factor_1 (temperature):20.0 | Factor_2 (bacteria presence):1.0 RAW_FILE_NAME=24; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=20°C Co-culture; Biological replicate=3 SUBJECT_SAMPLE_FACTORS - 27 Factor_1 (temperature):20.0 | Factor_2 (bacteria presence):1.0 RAW_FILE_NAME=27; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=20°C Co-culture; Biological replicate=4 SUBJECT_SAMPLE_FACTORS - 30 Factor_1 (temperature):28.0 | Factor_2 (bacteria presence):1.0 RAW_FILE_NAME=30; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=28°C Co-culture; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 39 Factor_1 (temperature):28.0 | Factor_2 (bacteria presence):1.0 RAW_FILE_NAME=39; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=28°C Co-culture; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 42 Factor_1 (temperature):28.0 | Factor_2 (bacteria presence):1.0 RAW_FILE_NAME=42; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=28°C Co-culture; Biological replicate=3 SUBJECT_SAMPLE_FACTORS - 45 Factor_1 (temperature):28.0 | Factor_2 (bacteria presence):1.0 RAW_FILE_NAME=45; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=28°C Co-culture; Biological replicate=4 SUBJECT_SAMPLE_FACTORS - 48 Factor_1 (temperature):14.0 | Factor_2 (bacteria presence):0.0 RAW_FILE_NAME=48; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=14°C Axenic; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 51 Factor_1 (temperature):14.0 | Factor_2 (bacteria presence):0.0 RAW_FILE_NAME=51; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=14°C Axenic; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 54 Factor_1 (temperature):14.0 | Factor_2 (bacteria presence):0.0 RAW_FILE_NAME=54; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=14°C Axenic; Biological replicate=3 SUBJECT_SAMPLE_FACTORS - 57 Factor_1 (temperature):14.0 | Factor_2 (bacteria presence):0.0 RAW_FILE_NAME=57; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=14°C Axenic; Biological replicate=4 SUBJECT_SAMPLE_FACTORS - 60 Factor_1 (temperature):20.0 | Factor_2 (bacteria presence):0.0 RAW_FILE_NAME=60; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=20°C Axenic; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 63 Factor_1 (temperature):20.0 | Factor_2 (bacteria presence):0.0 RAW_FILE_NAME=63; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=20°C Axenic; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 66 Factor_1 (temperature):20.0 | Factor_2 (bacteria presence):0.0 RAW_FILE_NAME=66; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=20°C Axenic; Biological replicate=3 SUBJECT_SAMPLE_FACTORS - 69 Factor_1 (temperature):20.0 | Factor_2 (bacteria presence):0.0 RAW_FILE_NAME=69; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=20°C Axenic; Biological replicate=4 SUBJECT_SAMPLE_FACTORS - 72 Factor_1 (temperature):28.0 | Factor_2 (bacteria presence):0.0 RAW_FILE_NAME=72; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=28°C Axenic; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 75 Factor_1 (temperature):28.0 | Factor_2 (bacteria presence):0.0 RAW_FILE_NAME=75; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=28°C Axenic; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 78 Factor_1 (temperature):28.0 | Factor_2 (bacteria presence):0.0 RAW_FILE_NAME=78; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=28°C Axenic; Biological replicate=3 SUBJECT_SAMPLE_FACTORS - 81 Factor_1 (temperature):28.0 | Factor_2 (bacteria presence):0.0 RAW_FILE_NAME=81; Dataset type=Endometabolome; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment name=Temperature experiment; Sample descritpion=28°C Axenic; Biological replicate=4 #COLLECTION CO:COLLECTION_SUMMARY 320 mL of diatom cells were collected by filtering the culture onto CO:COLLECTION_SUMMARY 2.0-µm-pore-size PCTE membrane filters (MilliporeSigma Isopore). Filters were CO:COLLECTION_SUMMARY kept in 50 mL tubes (Falcon) and stored at -80oC until processing. CO:COLLECTION_PROTOCOL_FILENAME 2_Collection protocol__UGA_phytoplankton_Oct2022.docx CO:SAMPLE_TYPE Algae #TREATMENT TR:TREATMENT_SUMMARY Six treatments of a marine diatom strain Thalassiosira pseudonana CCMP1335 were TR:TREATMENT_SUMMARY prepared: treatments incubated axenically at either 14, 20, or 28 oC, and TR:TREATMENT_SUMMARY treatments co-cultured with a bacterial strain Ruegeria pomeroyi DSS-3 at the TR:TREATMENT_SUMMARY corresponding temperatures (four replicates for each). L1 media was used with TR:TREATMENT_SUMMARY NaH13CO3 as a source of bicarbonate. The diatom used for the co-cultured TR:TREATMENT_SUMMARY treatments was B12 stressed to emphasize the known co-existing system. The light TR:TREATMENT_SUMMARY cycle consisted of 16 h light (120 µmol photons m-2 s-1) and 8 h of dark. TR:TREATMENT_PROTOCOL_FILENAME 3_Treatment protocol__UGA_phytoplankton_Oct2022.docx #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Phytoplankton cells were removed from filters using a sonicator SLPe (Branson) SP:SAMPLEPREP_SUMMARY in ultra-pure water (Millipore), concentrated by a lyophilizer (Labconco), and SP:SAMPLEPREP_SUMMARY kept -80oC until further processing. The samples were mixed with 600 µL of 30 SP:SAMPLEPREP_SUMMARY mmol L-1 sodium phosphate buffer (18 mmol L-1 NaHPO4, 12 mmol L-1, pH 7.4) and SP:SAMPLEPREP_SUMMARY an internal standard of 2,2-dimethyl-2-silapentane-5-sulfonate-d6 (DSS, 1 mmol SP:SAMPLEPREP_SUMMARY L-1), vortexed at 4oC for 5 minutes, centrifuged at 20,800 rcf using an SP:SAMPLEPREP_SUMMARY ultracentrifuge 5417C (Eppendorf) at 4oC for 10 minutes, and supernatants were SP:SAMPLEPREP_SUMMARY transferred to 5-mm NMR tubes (NORELL). SP:SAMPLEPREP_PROTOCOL_FILENAME 4_Sample preparation protocol__UGA_phytoplankton_Oct2022.docx #ANALYSIS AN:ANALYSIS_TYPE NMR #NMR NM:INSTRUMENT_NAME Bruker NEO NM:INSTRUMENT_TYPE CW-NMR NM:NMR_EXPERIMENT_TYPE Other NM:SPECTROMETER_FREQUENCY 600 MHz NM:NMR_RESULTS_FILE ST002321_AN003788_Results.txt UNITS:Intensity #END