#METABOLOMICS WORKBENCH neo_009_20150909_9229471_mwtab.txt DATATRACK_ID:360 STUDY_ID:ST000241 ANALYSIS_ID:AN000374 PROJECT_ID:PR000194 VERSION 1 CREATED_ON 2016-09-17 #PROJECT PR:PROJECT_TITLE Cyclobutene- and cyclobutane-functionalized fatty acids as novel biochemical PR:PROJECT_TITLE of structure and function in HepG2 cells PR:PROJECT_TYPE Lipidomics PR:PROJECT_SUMMARY Five analogues of OA (18:1cis9) or elaidic acid (18:1trans9) replacing the PR:PROJECT_SUMMARY with a four-membered carbocycle were evaluated in HepG2 cells, which are PR:PROJECT_SUMMARY models for hepatocytes. In order to assess whether or not the novel analogues PR:PROJECT_SUMMARY incorporated into complex lipids, cells were treated with compond and then PR:PROJECT_SUMMARY extracted. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry PR:PROJECT_SUMMARY was employed for the analysis of complex lipids. Data processing involved mass PR:PROJECT_SUMMARY chromatographic peak detection and deconvolution, isotopic peaks grouping, PR:PROJECT_SUMMARY and peak alignment. Significantly altered metabolites were defined by a fold PR:PROJECT_SUMMARY (FC) >2 and p<0.05. Principal component analysis (PCA) and hierarchical PR:PROJECT_SUMMARY analysis (HCA) of signature metabolites altered in compounds treated cells PR:PROJECT_SUMMARY to control were performed in the Metaboanalyst web portal PR:INSTITUTE University of Nebraska-Lincoln PR:DEPARTMENT Biochemistry PR:LABORATORY DiRusso Black FATTT Lab PR:LAST_NAME DiRusso PR:FIRST_NAME Concetta PR:ADDRESS Department of Biochemistry, University of Nebraska-Lincoln, N241 Beadle Center PR:ADDRESS Vine St. PR:EMAIL cdirusso2@unl.edu PR:PHONE 402-472-6504 or 402-613-9293 PR:FUNDING_SOURCE NIH #STUDY ST:STUDY_TITLE Cyclobutene- and cyclobutane-functionalized fatty acids as novel biochemical ST:STUDY_TITLE of structure and function in HepG2 cells ST:STUDY_TYPE Lipid analysis novel C18 fatty acid anologues in complex lipids ST:STUDY_SUMMARY Human hepatoma HepG2 cells (American Type Culture Collection; HB-8065) were ST:STUDY_SUMMARY in 75 ml tissue cell culture flasks in Eagle's minimal essential medium (EMEM) ST:STUDY_SUMMARY with 10% fetal bovine serum at 37°C in a humidified atmosphere with 5% CO2. ST:STUDY_SUMMARY treatment with fatty acids or analogues, the cells were seeded at a density of ST:STUDY_SUMMARY × 106 cells in a T25-cm2 flask for 24 h. Each FA or analogue was added to the ST:STUDY_SUMMARY medium as a fatty acid-bovine serum albumin (BSA) complex (2.5:1, FA:BSA) to ST:STUDY_SUMMARY the desired final concentration. The controls in these experiments were HepG2 ST:STUDY_SUMMARY with BSA alone. After 24 h treatment, the media was collected, cells were ST:STUDY_SUMMARY twice with PBS and cells were harvested for analysis. To each cell suspension ST:STUDY_SUMMARY to lipid extraction a standard mixture of 25 µg C15:0 PE, C17:0 PC and C71:1 ST:STUDY_SUMMARY was added as standards. Extraction of lipids was performed according to the ST:STUDY_SUMMARY method. For metabolomics analysis, the lipid extracts were resuspended in ST:STUDY_SUMMARY 2:1 (v/v).All analyses were carried out using an Agilent 1200 Series HPLC, ACE ST:STUDY_SUMMARY C8-300 column (2.1 x 100 mm) and linear gradient elution at a flow rate of 0.1 ST:STUDY_SUMMARY Mobile phase A and B consisted of 0.1% formic acid; 10 mM ammonium acetate in ST:STUDY_SUMMARY and 0.1% formic acid; 10 mM ammonium acetate in ACN/isopropanol (50/50; v/v), ST:STUDY_SUMMARY The injection volume was 4 µL. Separation of metabolites was achieved at the ST:STUDY_SUMMARY gradient: T=0 min: 30% B; T=1 min: 30% B; T=25 min: 100% B; T=45 min: 100% B; ST:STUDY_SUMMARY min: 30% B; and T=60 min: 30% B (re-equilibration). The HPLC system was ST:STUDY_SUMMARY coupled to a Bruker Soalrix 70 Hybrid FTMS instrument equipped with ST:STUDY_SUMMARY ionization source (ESI) (Bruker Daltonics). The system was controlled by HyStar ST:STUDY_SUMMARY software. MS data was collected with resolving power of 78,000 (at m/z 400) in ST:STUDY_SUMMARY or negative mode under following conditions: a capillary voltage of (+/-) 4,500 ST:STUDY_SUMMARY and an end plate offset of -500 V. The dry temperature was set at 180°C. Dry ST:STUDY_SUMMARY flow was maintained 4 L/min. Acquisition range was 244-1,800 m/z with 0.2 s ion ST:STUDY_SUMMARY time. LC-MS data was converted into mzXML format using CompassXport v. 3.0.6 ST:STUDY_SUMMARY processed by mzMine v.2.10 [25] or XCMS data analysis software. Data processing ST:STUDY_SUMMARY mass detection, chromatographic peak detection and deconvolution, isotopic ST:STUDY_SUMMARY grouping, normalization and peak alignment. Metabolite data were mean-centered ST:STUDY_SUMMARY unit-variance scaled to remove the offsets and adjust the importance of high ST:STUDY_SUMMARY low abundance metabolites to an equal level. Significantly altered metabolites ST:STUDY_SUMMARY defined by a fold change (FC) >2 and p<0.05. Principal component analysis (PCA) ST:STUDY_SUMMARY hierarchical clustering analysis (HCA) of signature metabolites altered in ST:STUDY_SUMMARY treated cells compared to control were performed in the Metaboanalyst web ST:STUDY_SUMMARY (www.metaboanalyst.ca). ST:INSTITUTE University of Nebraska - Lincoln ST:DEPARTMENT Biochemistry ST:LABORATORY DiRusso Black FATTT Lab ST:LAST_NAME DiRusso ST:FIRST_NAME Concetta ST:ADDRESS Department of Biochemistry, University of Nebraska-Lincoln, N241 Beadle Center ST:ADDRESS Vine St. ST:EMAIL cdirusso2@unl.edu ST:PHONE 402-472-6504 or 402-613-9293 ST:NUM_GROUPS 8 ST:TOTAL_SUBJECTS 24+24=48 ST:STUDY_COMMENTS 8 groups in triplicate ran in both negative and positive mode #SUBJECT SU:SUBJECT_TYPE Human cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC SU:CELL_STRAIN_DETAILS HepG2 SU:SUBJECT_COMMENTS NA SU:CELL_PRIMARY_IMMORTALIZED Immortalized SU:CELL_PASSAGE_NUMBER NA SU:CELL_COUNTS NA #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Control1 Concentration (uM):0 | Treatment_compound:BSA Sample_Data=BSA SUBJECT_SAMPLE_FACTORS - Control2 Concentration (uM):0 | Treatment_compound:BSA Sample_Data=BSA SUBJECT_SAMPLE_FACTORS - Control3 Concentration (uM):0 | Treatment_compound:BSA Sample_Data=BSA SUBJECT_SAMPLE_FACTORS - FAC1 Concentration (uM):500 | Treatment_compound:CB-cis Sample_Data=CB-cis, cis (1S*,4R*)-4-octyl-2-cyclobutene-1-octanoic acid SUBJECT_SAMPLE_FACTORS - FAC2 Concentration (uM):500 | Treatment_compound:CB-cis Sample_Data=CB-cis, cis (1S*,4R*)-4-octyl-2-cyclobutene-1-octanoic acid SUBJECT_SAMPLE_FACTORS - FAC3 Concentration (uM):500 | Treatment_compound:CB-cis Sample_Data=CB-cis, cis (1S*,4R*)-4-octyl-2-cyclobutene-1-octanoic acid SUBJECT_SAMPLE_FACTORS - FAT1 Concentration (uM):500 | Treatment_compound:CB-trans Sample_Data=CB-trans, trans (1R*,4R*)-4-octyl-2-cyclobutene-1-octanoic acid SUBJECT_SAMPLE_FACTORS - FAT2 Concentration (uM):500 | Treatment_compound:CB-trans Sample_Data=CB-trans, trans (1R*,4R*)-4-octyl-2-cyclobutene-1-octanoic acid SUBJECT_SAMPLE_FACTORS - FAT3 Concentration (uM):500 | Treatment_compound:CB-trans Sample_Data=CB-trans, trans (1R*,4R*)-4-octyl-2-cyclobutene-1-octanoic acid SUBJECT_SAMPLE_FACTORS - CKC1 Concentration (uM):500 | Treatment_compound:CK-cis Sample_Data=CK-cis, (1S*,3RS,4R*)-3-chloro-4-octyl-2-oxo cyclobutane-1-octanoic acid SUBJECT_SAMPLE_FACTORS - CKC2 Concentration (uM):500 | Treatment_compound:CK-cis Sample_Data=CK-cis, (1S*,3RS,4R*)-3-chloro-4-octyl-2-oxo cyclobutane-1-octanoic acid SUBJECT_SAMPLE_FACTORS - CKC3 Concentration (uM):500 | Treatment_compound:CK-cis Sample_Data=CK-cis, (1S*,3RS,4R*)-3-chloro-4-octyl-2-oxo cyclobutane-1-octanoic acid SUBJECT_SAMPLE_FACTORS - CKT1 Concentration (uM):500 | Treatment_compound:CK-trans Sample_Data=CK-trans, (1R*,3RS,4R*)-3-chloro-4-octyl-2-oxo cyclobutane-1-octanoic acid SUBJECT_SAMPLE_FACTORS - CKT2 Concentration (uM):500 | Treatment_compound:CK-trans Sample_Data=CK-trans, (1R*,3RS,4R*)-3-chloro-4-octyl-2-oxo cyclobutane-1-octanoic acid SUBJECT_SAMPLE_FACTORS - CKT3 Concentration (uM):500 | Treatment_compound:CK-trans Sample_Data=CK-trans, (1R*,3RS,4R*)-3-chloro-4-octyl-2-oxo cyclobutane-1-octanoic acid SUBJECT_SAMPLE_FACTORS - KC1 Concentration (uM):500 | Treatment_compound:K-cis Sample_Data=K-cis, cis-(1S*,4R*)- 4-octyl-2-oxocyclobutane-1-octanoic acid SUBJECT_SAMPLE_FACTORS - KC2 Concentration (uM):500 | Treatment_compound:K-cis Sample_Data=K-cis, cis-(1S*,4R*)- 4-octyl-2-oxocyclobutane-1-octanoic acid SUBJECT_SAMPLE_FACTORS - KC3 Concentration (uM):500 | Treatment_compound:K-cis Sample_Data=K-cis, cis-(1S*,4R*)- 4-octyl-2-oxocyclobutane-1-octanoic acid SUBJECT_SAMPLE_FACTORS - OA1 Concentration (uM):500 | Treatment_compound:Oleic acid Sample_Data=Oleic acid SUBJECT_SAMPLE_FACTORS - OA2 Concentration (uM):500 | Treatment_compound:Oleic acid Sample_Data=Oleic acid SUBJECT_SAMPLE_FACTORS - OA3 Concentration (uM):500 | Treatment_compound:Oleic acid Sample_Data=Oleic acid SUBJECT_SAMPLE_FACTORS - PA1 Concentration (uM):500 | Treatment_compound:Palmitic acid Sample_Data=Palmitic acid SUBJECT_SAMPLE_FACTORS - PA2 Concentration (uM):500 | Treatment_compound:Palmitic acid Sample_Data=Palmitic acid SUBJECT_SAMPLE_FACTORS - PA3 Concentration (uM):500 | Treatment_compound:Palmitic acid Sample_Data=Palmitic acid #COLLECTION CO:COLLECTION_SUMMARY - #TREATMENT TR:TREATMENT_SUMMARY Cells treated 24hr with 500 µM Fa, analogue or BSA control. Harvested and TR:TREATMENT_SUMMARY extracted and then resolved using Fourier Transform Ion Cyclotron Resonance TR:TREATMENT_SUMMARY Spectrometry (FTICR-MS) s TR:TREATMENT_PROTOCOL_ID CBC_treatment TR:TREATMENT_PROTOCOL_FILENAME See_Comments TR:TREATMENT_PROTOCOL_COMMENTS Human hepatoma HepG2 cells (American Type Culture Collection; HB-8065) were TR:TREATMENT_PROTOCOL_COMMENTS in 75 ml tissue cell culture flasks in Eagle's minimal essential medium (EMEM) TR:TREATMENT_PROTOCOL_COMMENTS with 10% fetal bovine serum at 37°C in a humidified atmosphere with 5% CO2. TR:TREATMENT_PROTOCOL_COMMENTS treatment with fatty acids or analogues, the cells were seeded at a density of TR:TREATMENT_PROTOCOL_COMMENTS × 106 cells in a T25-cm2 flask for 24 h. Each FA or analogue was added to the TR:TREATMENT_PROTOCOL_COMMENTS medium as a fatty acid-bovine serum albumin (BSA) complex (2.5:1, FA:BSA) to TR:TREATMENT_PROTOCOL_COMMENTS 500µM final concentration. The controls in these experiments were HepG2 cells TR:TREATMENT_PROTOCOL_COMMENTS BSA alone. After 24 h treatment, the media was collected, cells were rinsed TR:TREATMENT_PROTOCOL_COMMENTS with PBS and cells were harvested for complex lipid analysis. TR:TREATMENT_COMPOUND Fatty acid/BSA TR:TREATMENT_DOSE 500 µM (fatty acid) TR:TREATMENT_DOSEDURATION 24 hr TR:TREATMENT_VEHICLE PBS TR:CELL_GROWTH_CONTAINER 75 ml tissue cell culture flasks TR:CELL_MEDIA Eagle's minimal essential medium (EMEM) augmented with 10% fetal bovine serum TR:CELL_ENVIR_COND 37°C in a humidified atmosphere with 5% CO2 TR:CELL_HARVESTING Typsinize and scrape TR:CELL_MEDIA_LASTCHANGED 24 hr #SAMPLEPREP SP:SAMPLEPREP_SUMMARY - SP:SAMPLEPREP_PROTOCOL_COMMENTS For metabolomics analysis, the lipid extracts were resuspended in SP:SAMPLEPREP_PROTOCOL_COMMENTS 2:1 (v/v).All analyses were carried out using an Agilent 1200 Series HPLC, ACE SP:SAMPLEPREP_PROTOCOL_COMMENTS C8-300 column (2.1 x 100 mm) and linear gradient elution at a flow rate of 0.1 SP:SAMPLEPREP_PROTOCOL_COMMENTS Mobile phase A and B consisted of 0.1% formic acid; 10 mM ammonium acetate in SP:SAMPLEPREP_PROTOCOL_COMMENTS and 0.1% formic acid; 10 mM ammonium acetate in ACN/isopropanol (50/50; v/v), SP:SAMPLEPREP_PROTOCOL_COMMENTS The injection volume was 4 µL. Separation of metabolites was achieved at the SP:SAMPLEPREP_PROTOCOL_COMMENTS gradient: T=0 min: 30% B; T=1 min: 30% B; T=25 min: 100% B; T=45 min: 100% B; SP:SAMPLEPREP_PROTOCOL_COMMENTS min: 30% B; and T=60 min: 30% B (re-equilibration). The HPLC system was SP:SAMPLEPREP_PROTOCOL_COMMENTS coupled to a Bruker Soalrix 70 Hybrid FTMS instrument equipped with SP:SAMPLEPREP_PROTOCOL_COMMENTS ionization source (ESI) (Bruker Daltonics). The system was controlled by HyStar SP:SAMPLEPREP_PROTOCOL_COMMENTS software. MS data was collected with resolving power of 78,000 (at m/z 400) in SP:SAMPLEPREP_PROTOCOL_COMMENTS or negative mode under following conditions: a capillary voltage of (+/-) 4,500 SP:SAMPLEPREP_PROTOCOL_COMMENTS and an end plate offset of -500 V. The dry temperature was set at 180°C. Dry SP:SAMPLEPREP_PROTOCOL_COMMENTS flow was maintained 4 L/min. Acquisition range was 244-1,800 m/z with 0.2 s ion SP:SAMPLEPREP_PROTOCOL_COMMENTS time. LC-MS data was converted into mzXML format using CompassXport v. 3.0.6 SP:SAMPLEPREP_PROTOCOL_COMMENTS processed by mzMine v.2.10 [25] or XCMS data analysis software. Data processing SP:SAMPLEPREP_PROTOCOL_COMMENTS mass detection, chromatographic peak detection and deconvolution, isotopic SP:SAMPLEPREP_PROTOCOL_COMMENTS grouping, normalization and peak alignment. SP:EXTRACTION_METHOD Folche Lipid Extraction: Folch J, Lees M, Sloane-Stanley GH. A simple method SP:EXTRACTION_METHOD the isolation and purification of total lipids from animal tissues. J biol SP:EXTRACTION_METHOD 1957;226:497-509. SP:SAMPLE_SPIKING 25 µg C15:0 PE, C17:0 PC and C71:1 TAG added to 2 X 10e6 cells prior to lipid SP:CELL_TYPE HepG2 #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1200 CH:COLUMN_NAME ACE 5 C8-300 (100 x 2.1mm) CH:METHODS_FILENAME jac8lipidneg.m CH:CHROMATOGRAPHY_COMMENTS jalcms1lipneg.m CH:FLOW_RATE 0.1 mL/min CH:INTERNAL_STANDARD 25 g C15:0 PE, C17:0 PC and C71:1 TAG CH:SOLVENT_A 0.1% formic acid; 10 mM ammonium acetate in H2O CH:SOLVENT_B 0.1% formic acid; 10 mM ammonium acetate in ACN/isopropanol (50/50; v/v) CH:ANALYTICAL_TIME 60 min #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME DiRusso Black FATTT Lab AN:SOFTWARE_VERSION Compass solariXcontrol v1.5.0 103 (February 28, 2011) AN:DETECTOR_TYPE msDetector AN:DATA_FORMAT *.baf #MS MS:INSTRUMENT_NAME Bruker SolariX FT-ICR-MS MS:INSTRUMENT_TYPE FT-ICR-MS MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:DATAFORMAT *.d MS:MS_COMMENTS Results for unique - mode identifications with each fatty acid are available