#METABOLOMICS WORKBENCH neo_009_20200309_055659_mwtab.txt DATATRACK_ID:1937 STUDY_ID:ST001326 ANALYSIS_ID:AN002208 PROJECT_ID:PR000903 VERSION 1 CREATED_ON March 10, 2020, 4:58 pm #PROJECT PR:PROJECT_TITLE Untargeted lipidome changes in Chlamydomonas reinhardtii treated with small PR:PROJECT_TITLE molecules containing adamantane structures 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 lipidome, 2 structurally similar compounds were selected and compared with cells PR:PROJECT_SUMMARY grown without compounds as control for untargeted lipidome 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 lipidome changes in Chlamydomonas reinhardtii treated with small ST:STUDY_TITLE molecules containing adamantane structures 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 lipidome, 2 structurally similar compounds were selected and compared with cells ST:STUDY_SUMMARY grown without compounds as control for untargeted lipidome 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]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Nplus_A Group:Cont RAW_FILE_NAME=NplusA.mzXML SUBJECT_SAMPLE_FACTORS - Nplus_C Group:Cont RAW_FILE_NAME=NplusC.mzXML SUBJECT_SAMPLE_FACTORS - Nplus_B Group:Cont RAW_FILE_NAME=NplusB.mzXML SUBJECT_SAMPLE_FACTORS - 542_a Group:WD20542 RAW_FILE_NAME=542a.mzXML SUBJECT_SAMPLE_FACTORS - 542_b Group:WD20542 RAW_FILE_NAME=542b.mzXML SUBJECT_SAMPLE_FACTORS - 542_c Group:WD20542 RAW_FILE_NAME=542c.mzXML SUBJECT_SAMPLE_FACTORS - 067_b Group:WD20067 RAW_FILE_NAME=067b.mzXML SUBJECT_SAMPLE_FACTORS - 067_c Group:WD20067 RAW_FILE_NAME=067c.mzXML SUBJECT_SAMPLE_FACTORS - 067_a Group:WD20067 RAW_FILE_NAME=067a.mzXML #COLLECTION CO:COLLECTION_SUMMARY For lipid analysis, cells were grown in TAP media and treated with compounds at CO:COLLECTION_SUMMARY a final concentration of 5 µM. After 72h of growth, cells were harvested, media CO:COLLECTION_SUMMARY removed the biomass was lyophilized overnight at -50 °C. For lipid extraction CO:COLLECTION_SUMMARY dry biomass powder was accurately weighed 10 ± 0.5 milligram and resuspended in CO:COLLECTION_SUMMARY chloroform:methanol (2:1; 0.01% butylated hydroxytoluene (BHT)). The cell lysis CO:COLLECTION_SUMMARY was performed using bead mill (Qiagen TissueLyser LT, Qiagen Valencia, CA) for 5 CO:COLLECTION_SUMMARY min at 50 Hz power and further incubated for 30 min in a multi-tube vortexer CO:COLLECTION_SUMMARY (Fischer Scientific). Lower lipid phase was retrieved and transferred to a new CO:COLLECTION_SUMMARY clean tube and samples re-extracted. Both lipid phases were pooled together CO:COLLECTION_SUMMARY lipids were shaken first with 0.8% aqueous potassium chloride solution and then CO:COLLECTION_SUMMARY water. The organic phase was transferred to fresh tube and evaporated under the CO:COLLECTION_SUMMARY stream of nitrogen. The dried lipid samples were flushed with nitrogen and CO:COLLECTION_SUMMARY stored at -80 °C until analyzed on FT-MS. CO:SAMPLE_TYPE Algae 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 uM (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 lipids extraction. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For lipid analysis, cells were grown in TAP media and treated with compounds at SP:SAMPLEPREP_SUMMARY a final concentration of 5 µM. After 72h of growth, cells were harvested, media SP:SAMPLEPREP_SUMMARY removed the biomass was lyophilized overnight at -50 °C. For lipid extraction SP:SAMPLEPREP_SUMMARY dry biomass powder was accurately weighed 10 ± 0.5 milligram and resuspended in SP:SAMPLEPREP_SUMMARY chloroform:methanol (2:1; 0.01% butylated hydroxytoluene (BHT)). The cell lysis SP:SAMPLEPREP_SUMMARY was performed using bead mill (Qiagen TissueLyser LT, Qiagen Valencia, CA) for 5 SP:SAMPLEPREP_SUMMARY min at 50 Hz power and further incubated for 30 min in a multi-tube vortexer SP:SAMPLEPREP_SUMMARY (Fischer Scientific). Lower lipid phase was retrieved and transferred to a new SP:SAMPLEPREP_SUMMARY clean tube and samples re-extracted. Both lipid phases were pooled together SP:SAMPLEPREP_SUMMARY lipids were shaken first with 0.8% aqueous potassium chloride solution and then SP:SAMPLEPREP_SUMMARY water. The organic phase was transferred to fresh tube and evaporated under the SP:SAMPLEPREP_SUMMARY stream of nitrogen. The dried lipid samples were flushed with nitrogen and SP:SAMPLEPREP_SUMMARY stored at -80 °C until analyzed on FT-MS. SP:EXTRACTION_METHOD Bead beating #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The dried lipid samples were retrieved from -80 °C and dissolved into CH:CHROMATOGRAPHY_SUMMARY methanol:chloroform (1:1). FTICR-MS analysis was performed using an Agilent 1200 CH:CHROMATOGRAPHY_SUMMARY Series HPCL coupled to a 7.05 T Solarix FT-ICR (Bruker) equipped with an ESI CH:CHROMATOGRAPHY_SUMMARY source and controlled by HyStar v3.4.8.0 software. Five microliter of samples CH:CHROMATOGRAPHY_SUMMARY were injected onto an Ace 5 C8-300 column (2.1 x 100 mm) at a flow rate of 0.1 CH:CHROMATOGRAPHY_SUMMARY ml/min. Initial HPLC conditions consisted of 70% Solvent A (0.1% FA; 10 mM CH:CHROMATOGRAPHY_SUMMARY ammonium acetate in water) and 30% Solvent B (0.1% FA; 10 mM ammonium acetate in CH:CHROMATOGRAPHY_SUMMARY acetonitrile). Samples were eluted using the following gradient: 30% B held for CH:CHROMATOGRAPHY_SUMMARY 1 minute, increased to 100% B over 24 minutes and held for 20 minutes. The CH:CHROMATOGRAPHY_SUMMARY column was returned to initial conditions of 30% B over 2 minutes then allowed CH:CHROMATOGRAPHY_SUMMARY to re-equilibrate over 13 minutes. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1200 Series HPLC CH:COLUMN_NAME ACE 5 C8-300 (100 x 2.1mm) CH:FLOW_RATE 0.1 mL/min CH:SOLVENT_A 0.1% FA; 10 mM ammonium acetate in water CH:SOLVENT_B 0.1% FA; 10 mM ammonium acetate in acetonitrile CH:SAMPLE_INJECTION 5 uL #ANALYSIS AN:ANALYSIS_TYPE MS AN:DETECTOR_TYPE FT-ICR MS #MS MS:INSTRUMENT_NAME Bruker Solarix FT-ICR-MS MS:INSTRUMENT_TYPE FT-ICR MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Accurate MS analysis was performed on Bruker 7.05 T Solarix FT-ICR. Mass spectra MS:MS_COMMENTS were acquired in the positive ionization mode from m/z 150.58 to 2500 at a MS:MS_COMMENTS resolving power of 49,000. Capillary voltage was 4500 V with the end plate MS:MS_COMMENTS offset voltage set to -500 V. Drying temperature was set to 180 °C and with a MS:MS_COMMENTS drying gas flow rate of 4 L/min and nebulizer gas flow set to 1.0 bar. MS:MS_COMMENTS Immediately prior to acquisition the instrument was calibrated in the positive MS:MS_COMMENTS ESI mode using NaTFA clusters. Raw MS spectra were converted to mzXML format MS:MS_COMMENTS using CompassXport v. 3.0.6 and processed by Mzmine v 2.2.4. Feature detection MS:MS_COMMENTS was done on the centrioded data using a noise level of 1.0E06 intensity. Once MS:MS_COMMENTS the masses were detected then FTMS shoulder peak filtering was performed using MS:MS_COMMENTS Lorentzian extended algorithm and chromatograms were build using a min time span MS:MS_COMMENTS of 0.1 min, minimum height of 1.0E06 intensity and m/z tolerance was set at 10 MS:MS_COMMENTS ppm. Peak smoothing was performed using Savitzky-Golay algorithm with a filter MS:MS_COMMENTS width of 3. Chromatograms were deconvulated using Savitzky-Golay algorithm using MS:MS_COMMENTS minimum peak height set at 1.0E06 intensity. Finally all the peak lists were MS:MS_COMMENTS aligned using Join aligner method using mz tolerance of 10 ppm, RT tolerance of MS:MS_COMMENTS 0.5 min. The aligned peak list rows were filtered using duplicated peak list MS:MS_COMMENTS filter to obtain 3448 peaks. The missing peak data was recovered using the Gap MS:MS_COMMENTS filling method using the intensity tolerance of 0.1, mz tolerance of 10 ppm and MS:MS_COMMENTS RT tolerance of 0.5 min. RT correction was allowed at this stage. These peaks MS:MS_COMMENTS were identified using Lipid Search module within the mzMine 2. Potential lipids MS:MS_COMMENTS were annotated according to there accurate mass on MS1 level with following MS:MS_COMMENTS setting. Minimum number of carbon set to 28 and max to 60, max number of double MS:MS_COMMENTS bonds set to 10, m/z tolerance was set to 0.001 Da. Then search was performed MS:MS_COMMENTS with two ionization mode [M+H]+ and [M+NH4]+ to obtain identification on 353 MS:MS_COMMENTS peaks. Peak areas along with identification, m/z, retention time were exported MS:MS_COMMENTS out and brought into R environment for univariate and multivariate analysis. MS:MS_RESULTS_FILE ST001326_AN002208_Results.txt UNITS:intesity Has m/z:Yes Has RT:Yes RT units:Minutes #END