#METABOLOMICS WORKBENCH JKubanek_20210715_060800 DATATRACK_ID:2746 STUDY_ID:ST001880 ANALYSIS_ID:AN003046 PROJECT_ID:PR001186 VERSION 1 CREATED_ON July 21, 2021, 1:17 pm #PROJECT PR:PROJECT_TITLE Predator Cues Target Signaling Pathways in Toxic Algal Metabolome PR:PROJECT_TYPE Metabolomics to elucidate signaling pathway PR:PROJECT_SUMMARY Early detection of predators is critical to the survival of all living PR:PROJECT_SUMMARY organisms. For phytoplankton, recognition and response to chemical cues from PR:PROJECT_SUMMARY predators, as evidence of predation risk, is particularly crucial. The PR:PROJECT_SUMMARY phytoplankton Alexandrium minutum upregulates its toxicity when exposed to PR:PROJECT_SUMMARY copepodamides, a suite of compounds released by copepod predators. However, how PR:PROJECT_SUMMARY A. minutum perceives these predatory cues and what metabolic pathways are PR:PROJECT_SUMMARY involved in initiating toxin induction remains unknown. In this study LC/MS and PR:PROJECT_SUMMARY NMR-based metabolomics uncovered subtle physiological responses of A. minutum to PR:PROJECT_SUMMARY copepodamides, including dysregulation of valine biosynthesis and enhancement of PR:PROJECT_SUMMARY butanoate metabolism and arginine biosynthesis. While we have yet to identify a PR:PROJECT_SUMMARY chemoreceptor directly activated by copepod cues, based on the results of PR:PROJECT_SUMMARY inhibition experiments detection of copepodamides appears to disrupt the PR:PROJECT_SUMMARY activity of serine/threonine phosphatases leading to increased jasmonic acid PR:PROJECT_SUMMARY biosynthesis and signaling, which leads to amplified gonyautoxin biosynthesis in PR:PROJECT_SUMMARY A. minutum. This study is an important step toward a better understanding of PR:PROJECT_SUMMARY chemosensory ecology of predator-prey interactions in phytoplankton. PR:INSTITUTE Georgia Institute of Technology PR:DEPARTMENT School of Biological Sciences, School of Chemistry and Biochemistry, Center for PR:DEPARTMENT Microbial Dynamics and Infection, Parker H. Petit Institute for Bioengineering PR:DEPARTMENT and Bioscience PR:LABORATORY Kubanek Lab PR:LAST_NAME Brown PR:FIRST_NAME Emily PR:ADDRESS 950 Atlantic Dr Atlanta, GA, 30332, USA PR:EMAIL julia.kubanek@biosci.gatech.edu PR:PHONE 404-894-8424 PR:PROJECT_COMMENTS This study has 3 parts: 2 NMR (polar and non-polar metabolites) and MS PR:CONTRIBUTORS Emily R. Brown, Sam G. Moore, David A. Gaul, and Julia Kubanek #STUDY ST:STUDY_TITLE NMR Predator Cues Target Signaling Pathways in Toxic Algal Metabolome (Polar ST:STUDY_TITLE metabolites) ST:STUDY_TYPE 1H NMR Metabolomics to elucidate signaling pathway ST:STUDY_SUMMARY Metabolomics investigation of the phytoplankton Alexandrium minutum with and ST:STUDY_SUMMARY without copepod cues in order to explore cell signaling involved in toxin ST:STUDY_SUMMARY induction. ST:INSTITUTE Georgia Institute of Technology ST:DEPARTMENT School of Biological Sciences, School of Chemistry and Biochemistry, Center for ST:DEPARTMENT Microbial Dynamics and Infection, Parker H. Petit Institute for Bioengineering ST:DEPARTMENT and Bioscience ST:LABORATORY Kubanek Lab ST:LAST_NAME Brown ST:FIRST_NAME Emily ST:ADDRESS 950 Atlantic Dr Atlanta, Georgia USA 30332 ST:EMAIL julia.kubanek@biosci.gatech.edu ST:PHONE 404-894-8424 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 40 ST:STUDY_COMMENTS Part 1 of 3. This part includes NMR analysis of polar metabolites using oPLSDA. ST:STUDY_COMMENTS Parts 2 and 3 inlcude NMR analysis of nonpolar metabolites and the corresponding ST:STUDY_COMMENTS mass spectrometry metabolomics for both polar and non-polar metabolites. #SUBJECT SU:SUBJECT_TYPE Other organism SU:SUBJECT_SPECIES Alexandrium minutum SU:TAXONOMY_ID 39455 SU:GENOTYPE_STRAIN CCMP 113 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 - Control 1 Treatment:Control RAW_FILE_NAME=Control 1; Cellular_toxicity (fmol)=0.3; Cells_per_mL=20547; Cells_in_polar_NMR_analysis=3821742; Polar_NMR_solvent_volume (uL)=224; Cells_in_polar_MS_analysis=1273914; Polar_MS_solvent_volume (uL)=280 SUBJECT_SAMPLE_FACTORS - Control 2 Treatment:Control RAW_FILE_NAME=Control 2; Cellular_toxicity (fmol)=0.36; Cells_per_mL=20299; Cells_in_polar_NMR_analysis=3775614; Polar_NMR_solvent_volume (uL)=222; Cells_in_polar_MS_analysis=1258538; Polar_MS_solvent_volume (uL)=277 SUBJECT_SAMPLE_FACTORS - Control 3 Treatment:Control RAW_FILE_NAME=Control 3; Cellular_toxicity (fmol)=0.4; Cells_per_mL=20670; Cells_in_polar_NMR_analysis=3844620; Polar_NMR_solvent_volume (uL)=226; Cells_in_polar_MS_analysis=1281540; Polar_MS_solvent_volume (uL)=282 SUBJECT_SAMPLE_FACTORS - Control 4 Treatment:Control RAW_FILE_NAME=Control 4; Cellular_toxicity (fmol)=0.45; Cells_per_mL=20299; Cells_in_polar_NMR_analysis=3775614; Polar_NMR_solvent_volume (uL)=222; Cells_in_polar_MS_analysis=1258538; Polar_MS_solvent_volume (uL)=277 SUBJECT_SAMPLE_FACTORS - Control 5 Treatment:Control RAW_FILE_NAME=Control 5; Cellular_toxicity (fmol)=0.29; Cells_per_mL=20670; Cells_in_polar_NMR_analysis=3844620; Polar_NMR_solvent_volume (uL)=226; Cells_in_polar_MS_analysis=1281540; Polar_MS_solvent_volume (uL)=282 SUBJECT_SAMPLE_FACTORS - Control 6 Treatment:Control RAW_FILE_NAME=Control 6; Cellular_toxicity (fmol)=0.46; Cells_per_mL=21165; Cells_in_polar_NMR_analysis=3936690; Polar_NMR_solvent_volume (uL)=231; Cells_in_polar_MS_analysis=1312230; Polar_MS_solvent_volume (uL)=289 SUBJECT_SAMPLE_FACTORS - Control 7 Treatment:Control RAW_FILE_NAME=Control 7; Cellular_toxicity (fmol)=0.43; Cells_per_mL=20299; Cells_in_polar_NMR_analysis=3775614; Polar_NMR_solvent_volume (uL)=222; Cells_in_polar_MS_analysis=1258538; Polar_MS_solvent_volume (uL)=277 SUBJECT_SAMPLE_FACTORS - Control 8 Treatment:Control RAW_FILE_NAME=Control 8; Cellular_toxicity (fmol)=0.41; Cells_per_mL=20794; Cells_in_polar_NMR_analysis=3867684; Polar_NMR_solvent_volume (uL)=227; Cells_in_polar_MS_analysis=1289228; Polar_MS_solvent_volume (uL)=284 SUBJECT_SAMPLE_FACTORS - Control 9 Treatment:Control RAW_FILE_NAME=Control 9; Cellular_toxicity (fmol)=0.34; Cells_per_mL=20423; Cells_in_polar_NMR_analysis=3798678; Polar_NMR_solvent_volume (uL)=223; Cells_in_polar_MS_analysis=1266226; Polar_MS_solvent_volume (uL)=279 SUBJECT_SAMPLE_FACTORS - Control 10 Treatment:Control RAW_FILE_NAME=Control 10; Cellular_toxicity (fmol)=0.34; Cells_per_mL=20670; Cells_in_polar_NMR_analysis=3844620; Polar_NMR_solvent_volume (uL)=226; Cells_in_polar_MS_analysis=1281540; Polar_MS_solvent_volume (uL)=282 SUBJECT_SAMPLE_FACTORS - Control 11 Treatment:Control RAW_FILE_NAME=Control 11; Cellular_toxicity (fmol)=0.4; Cells_per_mL=19433; Cells_in_polar_NMR_analysis=3614538; Polar_NMR_solvent_volume (uL)=212; Cells_in_polar_MS_analysis=1204846; Polar_MS_solvent_volume (uL)=265 SUBJECT_SAMPLE_FACTORS - Control 12 Treatment:Control RAW_FILE_NAME=Control 12; Cellular_toxicity (fmol)=0.43; Cells_per_mL=20423; Cells_in_polar_NMR_analysis=3798678; Polar_NMR_solvent_volume (uL)=223; Cells_in_polar_MS_analysis=1266226; Polar_MS_solvent_volume (uL)=279 SUBJECT_SAMPLE_FACTORS - Control 13 Treatment:Control RAW_FILE_NAME=N/A; Cellular_toxicity (fmol)=0.31; Cells_per_mL=20299; Cells_in_polar_NMR_analysis=3775614; Polar_NMR_solvent_volume (uL)=222; Cells_in_polar_MS_analysis=1258538; Polar_MS_solvent_volume (uL)=277 SUBJECT_SAMPLE_FACTORS - Control 14 Treatment:Control RAW_FILE_NAME=Control 14; Cellular_toxicity (fmol)=0.36; Cells_per_mL=20175; Cells_in_polar_NMR_analysis=3752550; Polar_NMR_solvent_volume (uL)=220; Cells_in_polar_MS_analysis=1250850; Polar_MS_solvent_volume (uL)=275 SUBJECT_SAMPLE_FACTORS - Control 15 Treatment:Control RAW_FILE_NAME=Control 15; Cellular_toxicity (fmol)=0.37; Cells_per_mL=20052; Cells_in_polar_NMR_analysis=3729672; Polar_NMR_solvent_volume (uL)=219; Cells_in_polar_MS_analysis=1243224; Polar_MS_solvent_volume (uL)=274 SUBJECT_SAMPLE_FACTORS - Control 16 Treatment:Control RAW_FILE_NAME=Control 16; Cellular_toxicity (fmol)=0.43; Cells_per_mL=20670; Cells_in_polar_NMR_analysis=3844620; Polar_NMR_solvent_volume (uL)=226; Cells_in_polar_MS_analysis=1281540; Polar_MS_solvent_volume (uL)=282 SUBJECT_SAMPLE_FACTORS - Control 17 Treatment:Control RAW_FILE_NAME=Control 17; Cellular_toxicity (fmol)=0.43; Cells_per_mL=21165; Cells_in_polar_NMR_analysis=3936690; Polar_NMR_solvent_volume (uL)=231; Cells_in_polar_MS_analysis=1312230; Polar_MS_solvent_volume (uL)=289 SUBJECT_SAMPLE_FACTORS - Control 18 Treatment:Control RAW_FILE_NAME=Control 18; Cellular_toxicity (fmol)=0.34; Cells_per_mL=21165; Cells_in_polar_NMR_analysis=3936690; Polar_NMR_solvent_volume (uL)=231; Cells_in_polar_MS_analysis=1312230; Polar_MS_solvent_volume (uL)=289 SUBJECT_SAMPLE_FACTORS - Control 19 Treatment:Control RAW_FILE_NAME=Control 19; Cellular_toxicity (fmol)=0.41; Cells_per_mL=20547; Cells_in_polar_NMR_analysis=3821742; Polar_NMR_solvent_volume (uL)=224; Cells_in_polar_MS_analysis=1273914; Polar_MS_solvent_volume (uL)=280 SUBJECT_SAMPLE_FACTORS - Control 20 Treatment:Control RAW_FILE_NAME=Control 20; Cellular_toxicity (fmol)=0.35; Cells_per_mL=19309; Cells_in_polar_NMR_analysis=3591474; Polar_NMR_solvent_volume (uL)=211; Cells_in_polar_MS_analysis=1197158; Polar_MS_solvent_volume (uL)=264 SUBJECT_SAMPLE_FACTORS - Copepodamide 1 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 1; Cellular_toxicity (fmol)=0.95; Cells_per_mL=19804; Cells_in_polar_NMR_analysis=3683544; Polar_NMR_solvent_volume (uL)=216; Cells_in_polar_MS_analysis=1227848; Polar_MS_solvent_volume (uL)=270 SUBJECT_SAMPLE_FACTORS - Copepodamide 2 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 2; Cellular_toxicity (fmol)=0.78; Cells_per_mL=19309; Cells_in_polar_NMR_analysis=3591474; Polar_NMR_solvent_volume (uL)=211; Cells_in_polar_MS_analysis=1197158; Polar_MS_solvent_volume (uL)=264 SUBJECT_SAMPLE_FACTORS - Copepodamide 3 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 3; Cellular_toxicity (fmol)=0.64; Cells_per_mL=21165; Cells_in_polar_NMR_analysis=3936690; Polar_NMR_solvent_volume (uL)=231; Cells_in_polar_MS_analysis=1312230; Polar_MS_solvent_volume (uL)=289 SUBJECT_SAMPLE_FACTORS - Copepodamide 4 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 4; Cellular_toxicity (fmol)=1.02; Cells_per_mL=18938; Cells_in_polar_NMR_analysis=3522468; Polar_NMR_solvent_volume (uL)=207; Cells_in_polar_MS_analysis=1174156; Polar_MS_solvent_volume (uL)=228 SUBJECT_SAMPLE_FACTORS - Copepodamide 5 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 5; Cellular_toxicity (fmol)=0.69; Cells_per_mL=20299; Cells_in_polar_NMR_analysis=3775614; Polar_NMR_solvent_volume (uL)=222; Cells_in_polar_MS_analysis=1258538; Polar_MS_solvent_volume (uL)=277 SUBJECT_SAMPLE_FACTORS - Copepodamide 6 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 6; Cellular_toxicity (fmol)=0.74; Cells_per_mL=18938; Cells_in_polar_NMR_analysis=3522468; Polar_NMR_solvent_volume (uL)=207; Cells_in_polar_MS_analysis=1174156; Polar_MS_solvent_volume (uL)=258 SUBJECT_SAMPLE_FACTORS - Copepodamide 7 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 7; Cellular_toxicity (fmol)=0.7; Cells_per_mL=19061; Cells_in_polar_NMR_analysis=3545346; Polar_NMR_solvent_volume (uL)=208; Cells_in_polar_MS_analysis=1181782; Polar_MS_solvent_volume (uL)=260 SUBJECT_SAMPLE_FACTORS - Copepodamide 8 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 8; Cellular_toxicity (fmol)=0.74; Cells_per_mL=19433; Cells_in_polar_NMR_analysis=3614538; Polar_NMR_solvent_volume (uL)=212; Cells_in_polar_MS_analysis=1204846; Polar_MS_solvent_volume (uL)=265 SUBJECT_SAMPLE_FACTORS - Copepodamide 9 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 9; Cellular_toxicity (fmol)=0.89; Cells_per_mL=20299; Cells_in_polar_NMR_analysis=3775614; Polar_NMR_solvent_volume (uL)=222; Cells_in_polar_MS_analysis=1258538; Polar_MS_solvent_volume (uL)=277 SUBJECT_SAMPLE_FACTORS - Copepodamide 10 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 10; Cellular_toxicity (fmol)=0.85; Cells_per_mL=19928; Cells_in_polar_NMR_analysis=3706608; Polar_NMR_solvent_volume (uL)=218; Cells_in_polar_MS_analysis=1235536; Polar_MS_solvent_volume (uL)=272 SUBJECT_SAMPLE_FACTORS - Copepodamide 11 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 11; Cellular_toxicity (fmol)=0.86; Cells_per_mL=19928; Cells_in_polar_NMR_analysis=3706608; Polar_NMR_solvent_volume (uL)=218; Cells_in_polar_MS_analysis=1235536; Polar_MS_solvent_volume (uL)=272 SUBJECT_SAMPLE_FACTORS - Copepodamide 12 Treatment:Copepodamide RAW_FILE_NAME=N/A; Cellular_toxicity (fmol)=0.82; Cells_per_mL=19061; Cells_in_polar_NMR_analysis=3545346; Polar_NMR_solvent_volume (uL)=208; Cells_in_polar_MS_analysis=1181782; Polar_MS_solvent_volume (uL)=260 SUBJECT_SAMPLE_FACTORS - Copepodamide 13 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 13; Cellular_toxicity (fmol)=0.83; Cells_per_mL=18566; Cells_in_polar_NMR_analysis=3453276; Polar_NMR_solvent_volume (uL)=203; Cells_in_polar_MS_analysis=1151092; Polar_MS_solvent_volume (uL)=253 SUBJECT_SAMPLE_FACTORS - Copepodamide 14 Treatment:Copepodamide RAW_FILE_NAME=N/A; Cellular_toxicity (fmol)=0.71; Cells_per_mL=18319; Cells_in_polar_NMR_analysis=3407334; Polar_NMR_solvent_volume (uL)=200; Cells_in_polar_MS_analysis=1135778; Polar_MS_solvent_volume (uL)=250 SUBJECT_SAMPLE_FACTORS - Copepodamide 15 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 15; Cellular_toxicity (fmol)=0.87; Cells_per_mL=19433; Cells_in_polar_NMR_analysis=3614538; Polar_NMR_solvent_volume (uL)=212; Cells_in_polar_MS_analysis=1204846; Polar_MS_solvent_volume (uL)=265 SUBJECT_SAMPLE_FACTORS - Copepodamide 16 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 16; Cellular_toxicity (fmol)=0.59; Cells_per_mL=19433; Cells_in_polar_NMR_analysis=3614538; Polar_NMR_solvent_volume (uL)=212; Cells_in_polar_MS_analysis=1204846; Polar_MS_solvent_volume (uL)=265 SUBJECT_SAMPLE_FACTORS - Copepodamide 17 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 17; Cellular_toxicity (fmol)=0.71; Cells_per_mL=19185; Cells_in_polar_NMR_analysis=3568410; Polar_NMR_solvent_volume (uL)=209; Cells_in_polar_MS_analysis=1189470; Polar_MS_solvent_volume (uL)=262 SUBJECT_SAMPLE_FACTORS - Copepodamide 18 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 18; Cellular_toxicity (fmol)=0.62; Cells_per_mL=19928; Cells_in_polar_NMR_analysis=3706608; Polar_NMR_solvent_volume (uL)=218; Cells_in_polar_MS_analysis=1235536; Polar_MS_solvent_volume (uL)=272 SUBJECT_SAMPLE_FACTORS - Copepodamide 19 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 19; Cellular_toxicity (fmol)=0.88; Cells_per_mL=18442; Cells_in_polar_NMR_analysis=3430212; Polar_NMR_solvent_volume (uL)=201; Cells_in_polar_MS_analysis=1143404; Polar_MS_solvent_volume (uL)=252 SUBJECT_SAMPLE_FACTORS - Copepodamide 20 Treatment:Copepodamide RAW_FILE_NAME=Copepodamide 20; Cellular_toxicity (fmol)=0.6; Cells_per_mL=18814; Cells_in_polar_NMR_analysis=3499404; Polar_NMR_solvent_volume (uL)=205; Cells_in_polar_MS_analysis=1166468; Polar_MS_solvent_volume (uL)=257 #COLLECTION CO:COLLECTION_SUMMARY Alexandrium minutum cells were collected by vacuum filtration onto GF/F filters CO:COLLECTION_SUMMARY and quenched with liquid nitrogen. Frozen cells with filters were stored in foil CO:COLLECTION_SUMMARY (previously muffled for 3 h at 450 °C) at -80 °C until extraction. CO:COLLECTION_PROTOCOL_FILENAME Predator Cues Target Signaling Pathways in Toxic Algal Metabolome_protocol.pdf CO:SAMPLE_TYPE Algae CO:COLLECTION_METHOD Filtration and Freeze CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Twice, first at the start of the experiment and again 24 h later, 1.5 mL of TR:TREATMENT_SUMMARY either 0.2 µM copepodamides (copepod cue) in DMSO (treatment, n=20) or DMSO TR:TREATMENT_SUMMARY alone (control, n=20) were added aseptically to 300 mL cultures of Alexandrium TR:TREATMENT_SUMMARY minutum resulting in a final concentration of 1 nM copepodamides. TR:TREATMENT_PROTOCOL_FILENAME Predator Cues Target Signaling Pathways in Toxic Algal Metabolome_protocol.pdf #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Polar extracts were dissolved at a concentration equivalent to 1.70 x 10^7 A. SP:SAMPLEPREP_SUMMARY minutum cells mL-1 in 90:10 H2O/D2O (99.96% atom D2O; Cambridge Isotope Labs) SP:SAMPLEPREP_SUMMARY with 0.2 mM phosphate buffer (pH 7.4) and 0.25 mM 3 (trimethylsilyl)propionic SP:SAMPLEPREP_SUMMARY 2,2,3,3d 4 acid (TMSP), as internal standard, for 1H NMR spectroscopy analysis. SP:SAMPLEPREP_SUMMARY The media blank extract was prepared in the smallest volume of solvent possible SP:SAMPLEPREP_SUMMARY (175 µL). Particulates were excluded by briefly centrifuging the samples and SP:SAMPLEPREP_SUMMARY then carefully transferring the extracts into 3 mm NMR tubes. The extracts were SP:SAMPLEPREP_SUMMARY analyzed using a Bruker Avance IIIHD 800 MHz NMR spectrometer equipped with a 3 SP:SAMPLEPREP_SUMMARY mm triple resonance broadband cryoprobe. Spectra for the polar extracts were SP:SAMPLEPREP_SUMMARY acquired using a 1D water presaturation (Bruker zgpr pulse sequence) with a SP:SAMPLEPREP_SUMMARY spectral width of 12 kHz centered at 3758.36 Hz, with a relaxation delay 3 s, SP:SAMPLEPREP_SUMMARY over 480 scans. SP:SAMPLEPREP_PROTOCOL_FILENAME Predator Cues Target Signaling Pathways in Toxic Algal Metabolome_protocol.pdf SP:EXTRACT_STORAGE -80℃ #ANALYSIS AN:ANALYSIS_TYPE NMR #NMR NM:INSTRUMENT_NAME Bruker Avance IIIHD NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 800 MHz NM:NMR_PROBE 3 mm triple resonance broadband cryoprobe NM:NMR_SOLVENT 90:10 H2O/D2O (99.96% atom D2O; Cambridge Isotope Labs) with 0.2 mM phosphate NM:NMR_SOLVENT buffer (pH 7.4) and 0.25 mM 3-(trimethylsilyl)propionic-2,2,3,3d4-acid NM:NMR_TUBE_SIZE 3 mm NM:PULSE_SEQUENCE zgpr NM:NUMBER_OF_SCANS 480 NM:RELAXATION_DELAY 3s NM:SPECTRAL_WIDTH 12 kHz NM:BASELINE_CORRECTION_METHOD Spline NM:BINNED_INCREMENT 0.005 ppm NM:NMR_RESULTS_FILE Preprocessed_binned_polar_NMRmetabolomics.txt UNITS:area under the curve #END