#METABOLOMICS WORKBENCH JKubanek_20210720_085504_mwtab.txt DATATRACK_ID:2760 STUDY_ID:ST001896 ANALYSIS_ID:AN003077 PROJECT_ID:000000 VERSION 1 CREATED_ON July 20, 2021, 9:07 am #PROJECT PR:PROJECT_TITLE Differentiating toxic and nontoxic congeneric harmful algae using the non-polar PR:PROJECT_TITLE metabolome PR:PROJECT_TYPE Using metabolomics to compare species PR:PROJECT_SUMMARY Metabolomics comparison of toxic and non-toxic species of phytoplankton from the PR:PROJECT_SUMMARY genus Alexandrium.This study was carried out using 2 pairwise experiments, A. PR:PROJECT_SUMMARY catenella compared to A. tamarense (Experiment 1) and A. pacificum compared to PR:PROJECT_SUMMARY A. tamarense (Experiment 2). 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 (Experiment 1: A. catenella-A. tamarense & PR:PROJECT_COMMENTS Experiment 2: A. pacificum-A. tamarense) and MS PR:CONTRIBUTORS Emily R. Brown, Sam G. Moore, David A. Gaul, and Julia Kubanek #STUDY ST:STUDY_TITLE NMR Differentiating toxic and nontoxic congeneric harmful algae using the ST:STUDY_TITLE non-polar metabolome (Experiment 2) ST:STUDY_TYPE 1H NMR Metabolomics to compare toxic and non-toxic species ST:STUDY_SUMMARY Metabolomics comparison of toxic and non-toxic species of phytoplankton from the ST:STUDY_SUMMARY genus Alexandrium.This study was carried out using 2 pairwise experiments, A. ST:STUDY_SUMMARY catenella compared to A. tamarense (Experiment 1) and A. pacificum compared to ST:STUDY_SUMMARY A. tamarense (Experiment 2). This study includes the NMR data from Experiment 2. 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 30 ST:STUDY_COMMENTS Part 2 of 3. This part includes NMR analysis of non-polar metabolites from ST:STUDY_COMMENTS Experiment 2 using oPLSDA. Parts 1 and 3 inlcude NMR analysis of non-polar ST:STUDY_COMMENTS metabolites from Experiment 1 and the corresponding mass spectrometry ST:STUDY_COMMENTS metabolomics for both Experiments 1 and 2. #SUBJECT SU:SUBJECT_TYPE Other organism SU:SUBJECT_SPECIES Alexandrium pacificum; Alexandrium tamarense SU:TAXONOMY_ID 1565494; 2926 SU:GENOTYPE_STRAIN CCMP 1493; CCMP 2023 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 - Apacificum 1 Treatment:Alexandrium_pacificum RAW_FILE_NAME=Apacificum 1; Cells_per_mL=17947; Number_of_cells_extracted=4468803; NMR_solvent_volume (uL)=276; MS_solvent_volume (uL)=984 SUBJECT_SAMPLE_FACTORS - Apacificum 2 Treatment:Alexandrium_pacificum RAW_FILE_NAME=Apacificum 2; Cells_per_mL=17947; Number_of_cells_extracted=4468803; NMR_solvent_volume (uL)=276; MS_solvent_volume (uL)=984 SUBJECT_SAMPLE_FACTORS - Apacificum 3 Treatment:Alexandrium_pacificum RAW_FILE_NAME=Apacificum 3; Cells_per_mL=14853; Number_of_cells_extracted=3698397; NMR_solvent_volume (uL)=229; MS_solvent_volume (uL)=814 SUBJECT_SAMPLE_FACTORS - Apacificum 4 Treatment:Alexandrium_pacificum RAW_FILE_NAME=Apacificum 4; Cells_per_mL=16957; Number_of_cells_extracted=4222293; NMR_solvent_volume (uL)=261; MS_solvent_volume (uL)=929 SUBJECT_SAMPLE_FACTORS - Apacificum 5 Treatment:Alexandrium_pacificum RAW_FILE_NAME=Apacificum 5; Cells_per_mL=15967; Number_of_cells_extracted=3975783; NMR_solvent_volume (uL)=246; MS_solvent_volume (uL)=875 SUBJECT_SAMPLE_FACTORS - Apacificum 6 Treatment:Alexandrium_pacificum RAW_FILE_NAME=none; Cells_per_mL=16462; Number_of_cells_extracted=4099038; NMR_solvent_volume (uL)=253; MS_solvent_volume (uL)=902 SUBJECT_SAMPLE_FACTORS - Apacificum 7 Treatment:Alexandrium_pacificum RAW_FILE_NAME=none; Cells_per_mL=15224; Number_of_cells_extracted=3790776; NMR_solvent_volume (uL)=234; MS_solvent_volume (uL)=834 SUBJECT_SAMPLE_FACTORS - Apacificum 8 Treatment:Alexandrium_pacificum RAW_FILE_NAME=Apacificum 8; Cells_per_mL=16710; Number_of_cells_extracted=4160790; NMR_solvent_volume (uL)=257; MS_solvent_volume (uL)=916 SUBJECT_SAMPLE_FACTORS - Apacificum 9 Treatment:Alexandrium_pacificum RAW_FILE_NAME=Apacificum 9; Cells_per_mL=15348; Number_of_cells_extracted=3821652; NMR_solvent_volume (uL)=236; MS_solvent_volume (uL)=841 SUBJECT_SAMPLE_FACTORS - Apacificum 10 Treatment:Alexandrium_pacificum RAW_FILE_NAME=Apacificum 10; Cells_per_mL=15348; Number_of_cells_extracted=3821652; NMR_solvent_volume (uL)=236; MS_solvent_volume (uL)=841 SUBJECT_SAMPLE_FACTORS - Apacificum 11 Treatment:Alexandrium_pacificum RAW_FILE_NAME=Apacificum 11; Cells_per_mL=18071; Number_of_cells_extracted=4499679; NMR_solvent_volume (uL)=278; MS_solvent_volume (uL)=990 SUBJECT_SAMPLE_FACTORS - Apacificum 12 Treatment:Alexandrium_pacificum RAW_FILE_NAME=Apacificum 12; Cells_per_mL=15472; Number_of_cells_extracted=3852528; NMR_solvent_volume (uL)=238; MS_solvent_volume (uL)=848 SUBJECT_SAMPLE_FACTORS - Apacificum 13 Treatment:Alexandrium_pacificum RAW_FILE_NAME=Apacificum 13; Cells_per_mL=15224; Number_of_cells_extracted=3790776; NMR_solvent_volume (uL)=234; MS_solvent_volume (uL)=834 SUBJECT_SAMPLE_FACTORS - Apacificum 14 Treatment:Alexandrium_pacificum RAW_FILE_NAME=Apacificum 14; Cells_per_mL=14729; Number_of_cells_extracted=3667521; NMR_solvent_volume (uL)=227; MS_solvent_volume (uL)=807 SUBJECT_SAMPLE_FACTORS - Apacificum 15 Treatment:Alexandrium_pacificum RAW_FILE_NAME=Apacificum 15; Cells_per_mL=14977; Number_of_cells_extracted=3729273; NMR_solvent_volume (uL)=230; MS_solvent_volume (uL)=821 SUBJECT_SAMPLE_FACTORS - Atamarense 1 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 1; Cells_per_mL=18319; Number_of_cells_extracted=4561431; NMR_solvent_volume (uL)=282; MS_solvent_volume (uL)=1004 SUBJECT_SAMPLE_FACTORS - Atamarense 2 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 2; Cells_per_mL=17947; Number_of_cells_extracted=4468803; NMR_solvent_volume (uL)=276; MS_solvent_volume (uL)=984 SUBJECT_SAMPLE_FACTORS - Atamarense 3 Treatment:Alexandrium_tamarense RAW_FILE_NAME=none; Cells_per_mL=17947; Number_of_cells_extracted=4468803; NMR_solvent_volume (uL)=276; MS_solvent_volume (uL)=984 SUBJECT_SAMPLE_FACTORS - Atamarense 4 Treatment:Alexandrium_tamarense RAW_FILE_NAME=none; Cells_per_mL=17947; Number_of_cells_extracted=4468803; NMR_solvent_volume (uL)=276; MS_solvent_volume (uL)=984 SUBJECT_SAMPLE_FACTORS - Atamarense 5 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 5; Cells_per_mL=17081; Number_of_cells_extracted=4253169; NMR_solvent_volume (uL)=263; MS_solvent_volume (uL)=936 SUBJECT_SAMPLE_FACTORS - Atamarense 6 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 6; Cells_per_mL=17576; Number_of_cells_extracted=4376424; NMR_solvent_volume (uL)=270; MS_solvent_volume (uL)=963 SUBJECT_SAMPLE_FACTORS - Atamarense 7 Treatment:Alexandrium_tamarense RAW_FILE_NAME=none; Cells_per_mL=17947; Number_of_cells_extracted=4468803; NMR_solvent_volume (uL)=276; MS_solvent_volume (uL)=984 SUBJECT_SAMPLE_FACTORS - Atamarense 8 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 8; Cells_per_mL=16462; Number_of_cells_extracted=4099038; NMR_solvent_volume (uL)=253; MS_solvent_volume (uL)=902 SUBJECT_SAMPLE_FACTORS - Atamarense 9 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 9; Cells_per_mL=16957; Number_of_cells_extracted=4222293; NMR_solvent_volume (uL)=261; MS_solvent_volume (uL)=929 SUBJECT_SAMPLE_FACTORS - Atamarense 10 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 10; Cells_per_mL=16462; Number_of_cells_extracted=4099038; NMR_solvent_volume (uL)=253; MS_solvent_volume (uL)=902 SUBJECT_SAMPLE_FACTORS - Atamarense 11 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 11; Cells_per_mL=15596; Number_of_cells_extracted=3883404; NMR_solvent_volume (uL)=240; MS_solvent_volume (uL)=855 SUBJECT_SAMPLE_FACTORS - Atamarense 12 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 12; Cells_per_mL=17081; Number_of_cells_extracted=4253169; NMR_solvent_volume (uL)=263; MS_solvent_volume (uL)=936 SUBJECT_SAMPLE_FACTORS - Atamarense 13 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 13; Cells_per_mL=16462; Number_of_cells_extracted=4099038; NMR_solvent_volume (uL)=253; MS_solvent_volume (uL)=902 SUBJECT_SAMPLE_FACTORS - Atamarense 14 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 14; Cells_per_mL=15101; Number_of_cells_extracted=3760149; NMR_solvent_volume (uL)=232; MS_solvent_volume (uL)=828 SUBJECT_SAMPLE_FACTORS - Atamarense 15 Treatment:Alexandrium_tamarense RAW_FILE_NAME=none; Cells_per_mL=17205; Number_of_cells_extracted=4284045; NMR_solvent_volume (uL)=265; MS_solvent_volume (uL)=943 #COLLECTION CO:COLLECTION_SUMMARY Alexandrium cells were collected by vacuum filtration onto GF/F filters and CO:COLLECTION_SUMMARY 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 Differentiating toxic and nontoxic congeneric harmful algae using the non-polar CO:COLLECTION_PROTOCOL_FILENAME metabolome_protocol.pdf CO:SAMPLE_TYPE Algae CO:COLLECTION_METHOD Filtration and Freeze CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Metabolomes of toxic versus non-toxic species were compared using the following TR:TREATMENT_SUMMARY experimental pairings: A. tamarense (n=15) with A. catenella (n=15) (Experiment TR:TREATMENT_SUMMARY 1) and A. tamarense (n=15) with A. pacificum (n=15) (Experiment 2). The same TR:TREATMENT_SUMMARY non-toxic strain of A. tamarense was used in both experiments but the two TR:TREATMENT_SUMMARY experiments were conducted separately, in different months, to make the TR:TREATMENT_SUMMARY experiment manageable based on availability of batches grown from stock TR:TREATMENT_SUMMARY cultures. For both experiments, Alexandrium spp. cultures at a cell density of TR:TREATMENT_SUMMARY 12,000 to 13,000 cells mL-1 were split into fifteen 300 mL subcultures of each TR:TREATMENT_SUMMARY species which grew for two days. At the end of each experiment, during TR:TREATMENT_SUMMARY harvesting, a 1.0 mL aliquot from each culture was preserved with Lugol’s TR:TREATMENT_SUMMARY solution to measure cell concentrations TR:TREATMENT_PROTOCOL_FILENAME Differentiating toxic and nontoxic congeneric harmful algae using the non-polar TR:TREATMENT_PROTOCOL_FILENAME metabolome_protocol.pdf TR:TREATMENT Species comparison #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Non polar extracts were dissolved with volumes calculated to account for SP:SAMPLEPREP_SUMMARY different cell densities at the time of harvesting, to a concentration SP:SAMPLEPREP_SUMMARY equivalent to 1.62 x 10^7 Alexandrium cells mL-1 in dimethyl sulfoxide d6 (99.9% SP:SAMPLEPREP_SUMMARY atom d6-DMSO; Cambridge Isotope Labs) containing 0.1% trimethylsilane (TMS) as SP:SAMPLEPREP_SUMMARY internal standard for 1H nuclear magnetic resonance (NMR) spectroscopy analysis. SP:SAMPLEPREP_SUMMARY The extracts were transferred to 3 mm NMR tubes. Extracts from Experiment 2 (A. SP:SAMPLEPREP_SUMMARY tamarense-A. pacificum) were analyzed using a Bruker Avance IIIHD 700 MHz NMR SP:SAMPLEPREP_SUMMARY spectrometer equipped with a 5 mm indirect broadband cryoprobe, because the 800 SP:SAMPLEPREP_SUMMARY MHz instrument was unavailable at the time of data acquisition. Spectra of SP:SAMPLEPREP_SUMMARY non-polar extracts from both experiments were acquired using a Bruker zg30 pulse SP:SAMPLEPREP_SUMMARY sequence 1H NMR experiment compiled from 320 scans. SP:SAMPLEPREP_PROTOCOL_FILENAME Differentiating toxic and nontoxic congeneric harmful algae using the non-polar SP:SAMPLEPREP_PROTOCOL_FILENAME 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 700 MHz NM:NMR_PROBE 5 mm indirect broadband cryoprobe NM:NMR_SOLVENT Dimethyl sulfoxide d6 (99.9% atom d6-DMSO; Cambridge Isotope Labs) containing NM:NMR_SOLVENT 0.1% trimethylsilane NM:NMR_TUBE_SIZE 3 mm NM:PULSE_SEQUENCE zg30 NM:NUMBER_OF_SCANS 320 NM:BASELINE_CORRECTION_METHOD Spline NM:BINNED_INCREMENT 0.005 ppm NM:NMR_RESULTS_FILE Preprocessed_binned_Experiment2_ApvAt_NMRmetabolomics.txt UNITS:area under the curve #END