#METABOLOMICS WORKBENCH neo_009_20170616_101623 DATATRACK_ID:978 STUDY_ID:ST000621 ANALYSIS_ID:AN000953 PROJECT_ID:PR000454 VERSION 1 CREATED_ON June 19, 2017, 1:45 pm #PROJECT PR:PROJECT_TITLE Untargeted metabolomic changes in Chlamydomonas reinhardtii treated with lipid PR:PROJECT_TITLE inducing small molecules PR:PROJECT_SUMMARY A study to investigate the effect of small molecule lipid inducing compounds PR:PROJECT_SUMMARY that leads to hyper accumulation of lipids in N replete cells of Chlamydomonas PR:PROJECT_SUMMARY reinhardtii. These compounds were identified through a high throughput screening PR:PROJECT_SUMMARY designed for that purpose. During that screening, we screened 43,783 compounds PR:PROJECT_SUMMARY and identified 367 primary hits. These 367 hits were further retested using a PR:PROJECT_SUMMARY 8-point dilution series (from 0.25 to 30 uM) and verified the activity of 250 PR:PROJECT_SUMMARY compounds that induce the hyper lipid accumulating phenotype in algae. Once the PR:PROJECT_SUMMARY hit compounds were identified and confirmed, we then performed extensive PR:PROJECT_SUMMARY chemoinformatics analysis to look for common scaffolds and identified several PR:PROJECT_SUMMARY common substructures. We then selected 15 top performing compounds from 5 PR:PROJECT_SUMMARY diverse structural groups and tested biochemical parameters such as growth, PR:PROJECT_SUMMARY lipid accumulating capacity, effect on photosynthetic rates, respiration rates, PR:PROJECT_SUMMARY oxygen consumption rates, analysis of different lipid species to quantify and PR:PROJECT_SUMMARY identify fatty acid species using GC-MS. To understand the global changes in the PR:PROJECT_SUMMARY metabolome, 2 structurally different compounds were selected and compared with PR:PROJECT_SUMMARY cells grown without compounds as control for untargeted metabolomics analysis. PR:INSTITUTE University of Nebraska-Lincoln PR:DEPARTMENT Biochemistry PR:LABORATORY FATTTLab PR:LAST_NAME Wase PR:FIRST_NAME Nishikant PR:ADDRESS 1901 Beadle Center, Vine Street, 1901 VINE STREET, Lincoln, NE, 68588-0664, USA PR:EMAIL nishikant.wase@gmail.com PR:PHONE 4023109931 PR:FUNDING_SOURCE NCESR-704, Nebraska Center for Energy Science Research; EPS-1004094 and 1264409, PR:FUNDING_SOURCE National Science Foundation ; NSF CBET : 1402896, National Science Foundation PR:CONTRIBUTORS Nishikant Wase, Jiri Adamec, Ron Cerny, Girish Rasineni, Paul N Black, Concetta PR:CONTRIBUTORS DiRusso #STUDY ST:STUDY_TITLE Untargeted metabolomic changes in Chlamydomonas reinhardtii treated with lipid ST:STUDY_TITLE inducing small molecules ST:STUDY_SUMMARY A study to investigate the effect of small molecule lipid inducing compounds ST:STUDY_SUMMARY that leads to hyper accumulation of lipids in N replete cells of Chlamydomonas ST:STUDY_SUMMARY reinhardtii. These compounds were identified through a high throughput screening ST:STUDY_SUMMARY designed for that purpose. During that screening, we screened 43,783 compounds ST:STUDY_SUMMARY and identified 367 primary hits. These 367 hits were further retested using a ST:STUDY_SUMMARY 8-point dilution series (from 0.25 to 30 uM) and verified the activity of 250 ST:STUDY_SUMMARY compounds that induce the hyper lipid accumulating phenotype in algae. Once the ST:STUDY_SUMMARY hit compounds were identified and confirmed, we then performed extensive ST:STUDY_SUMMARY chemoinformatics analysis to look for common scaffolds and identified several ST:STUDY_SUMMARY common substructures. We then selected 15 top performing compounds from 5 ST:STUDY_SUMMARY diverse structural groups and tested biochemical parameters such as growth, ST:STUDY_SUMMARY lipid accumulating capacity, effect on photosynthetic rates, respiration rates, ST:STUDY_SUMMARY oxygen consumption rates, analysis of different lipid species to quantify and ST:STUDY_SUMMARY identify fatty acid species using GC-MS. To understand the global changes in the ST:STUDY_SUMMARY metabolome, 2 structurally different compounds were selected and compared with ST:STUDY_SUMMARY cells grown without compounds as control for untargeted metabolomics analysis. ST:INSTITUTE University of Nebraska-Lincoln ST:DEPARTMENT Department of Biochemistry ST:LAST_NAME Wase ST:FIRST_NAME Nishikant ST:ADDRESS Department of Biochemistry, 1901 VINE STREET ST:EMAIL nishikant.wase@gmail.com ST:PHONE 4023109931 #SUBJECT SU:SUBJECT_TYPE Photosynthetic organism SU:SUBJECT_SPECIES Chlamydomonas reinhardtii SU:TAXONOMY_ID 3055 SU:GENOTYPE_STRAIN CC125 Wild type #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - CD06241603 Group:Control Treatment=DMSO SUBJECT_SAMPLE_FACTORS - CD06241601B Group:Control Treatment=DMSO SUBJECT_SAMPLE_FACTORS - CD06241602 Group:Control Treatment=DMSO SUBJECT_SAMPLE_FACTORS - CD06241619 Group:cmp_784 Treatment=5 uM Compound WD10784 SUBJECT_SAMPLE_FACTORS - CD06241620 Group:cmp_784 Treatment=5 uM Compound WD10784 SUBJECT_SAMPLE_FACTORS - CD06241621 Group:cmp_784 Treatment=5 uM Compound WD10784 SUBJECT_SAMPLE_FACTORS - CD06241608 Group:cmp_030 Treatment=5 uM Compound WD30030 SUBJECT_SAMPLE_FACTORS - CD06241609 Group:cmp_030 Treatment=5 uM Compound WD30030 SUBJECT_SAMPLE_FACTORS - CD06241607 Group:cmp_030 Treatment=5 uM Compound WD30030 #COLLECTION CO:COLLECTION_SUMMARY Triplicate cultures were grown and treated as indicated and harvested at 72 h. CO:COLLECTION_SUMMARY The harvested cells were freeze-dried and weighted 10 ± 0.5 mg dry powder. To CO:COLLECTION_SUMMARY extract the metabolites, 500 uL of 75% MeOH in 0.15 M NaCl was added and CO:COLLECTION_SUMMARY metabolites were extracted using bead beating 50 Hz for 5 min. The homogenate CO:COLLECTION_SUMMARY was sonicated in water bath for 5 min and then 300 uL of 0.15 M NaCl was added CO:COLLECTION_SUMMARY and then 1 mL of chloroform:methanol (1:2) with 0.01% BHT was added. Samples CO:COLLECTION_SUMMARY were homogenized and allowed to phase separate on ice for 30 mins. Finally tubes CO:COLLECTION_SUMMARY were centrifuged for 10 min to separate the polar and non-polar layer. Top polar CO:COLLECTION_SUMMARY layer was removed and an aliquot of 200 uL was used for analysis. Samples were CO:COLLECTION_SUMMARY evaporated to dryness in SpeedVac and metabolites were reconstituted in 0.03% CO:COLLECTION_SUMMARY formic acid in analytical-grade water. Debris was removed via centrifugation and CO:COLLECTION_SUMMARY the supernatant was subjected to UPLC-MS analysis. CO:SAMPLE_TYPE Photosynthetic organism CO:COLLECTION_METHOD Centrifugation from suspension culture. CO:COLLECTION_LOCATION FATTTLab Department of Biochemistry, Univ of Nebraska-Lincoln CO:STORAGE_CONDITIONS After harvest, cells were kept at -80 C until extraction. CO:COLLECTION_VIALS 2 mL Eppendorf tubes #TREATMENT TR:TREATMENT_SUMMARY For compound treatment, cells from mid-log phase culture were harvested, washed TR:TREATMENT_SUMMARY once with fresh sterile TAP media and inoculated at starting density of 5 x 105 TR:TREATMENT_SUMMARY cells/mL. Thus for the current experiment 3 different treatment conditions were TR:TREATMENT_SUMMARY generated. Cells without compound treatment were negative control for the lipid TR:TREATMENT_SUMMARY accumulation and 2 compounds were used to generate treatment conditions. All TR:TREATMENT_SUMMARY compounds were used at a final concentration of 5 M (this concentration was TR:TREATMENT_SUMMARY determined using a dose-response curve). For all cultures, 250 mL Erlenmeyer TR:TREATMENT_SUMMARY flasks with rubber stopper adopted to facilitate gas exchange were used and TR:TREATMENT_SUMMARY maintained in horizontal orbital shaking growing chamber (Innova 43; New TR:TREATMENT_SUMMARY Brunswick). At the end of 72h, cells were harvested, media was removed via TR:TREATMENT_SUMMARY centrifugation and biomass was stored at -80 C until metabolite extraction. TR:CELL_MEDIA Tris-Acetate Phoshphate (TAP) media. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Triplicate cultures were grown and treated as indicated and harvested at 72 h. SP:SAMPLEPREP_SUMMARY The harvested cells were freeze-dried and weighted 10 ± 0.5 mg dry powder. To SP:SAMPLEPREP_SUMMARY extract the metabolites, 500 uL of 75% MeOH in 0.15 M NaCl was added and SP:SAMPLEPREP_SUMMARY metabolites were extracted using bead beating 50 Hz for 5 min. The homogenate SP:SAMPLEPREP_SUMMARY was sonicated in water bath for 5 min and then 300 uL of 0.15 M NaCl was added SP:SAMPLEPREP_SUMMARY and then 1 mL of chloroform:methanol (1:2) with 0.01% BHT was added. Samples SP:SAMPLEPREP_SUMMARY were homogenized and allowed to phase separate on ice for 30 mins. Finally tubes SP:SAMPLEPREP_SUMMARY were centrifuged for 10 min to separate the polar and non-polar layer. Top polar SP:SAMPLEPREP_SUMMARY layer was removed and an aliquot of 200 uL was used for analysis. Samples were SP:SAMPLEPREP_SUMMARY evaporated to dryness in SpeedVac and metabolites were reconstituted in 0.03% SP:SAMPLEPREP_SUMMARY formic acid in analytical-grade water. Debris was removed via centrifugation and SP:SAMPLEPREP_SUMMARY the supernatant was subjected to UPLC-MS analysis SP:EXTRACTION_METHOD Bead beating #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1200 CH:COLUMN_NAME ACE 5 C18-300 (100 x 2.1mm) CH:FLOW_RATE 0.1 mL/min CH:SOLVENT_A 0.1% formic acid in water CH:SOLVENT_B 0.1% formic acid in MetOH CH:SAMPLE_INJECTION 8 uL #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Waters Synapt G2 S QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST000621_AN000953_Results.txt UNITS:intensity #END