Summary of Study ST000241

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000194. The data can be accessed directly via it's Project DOI: 10.21228/M89W2D This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST000241
Study TitleCyclobutene- and cyclobutane-functionalized fatty acids as novel biochemical probes of structure and function in HepG2 cells
Study TypeLipid analysis novel C18 fatty acid anologues in complex lipids
Study SummaryHuman hepatoma HepG2 cells (American Type Culture Collection; HB-8065) were grown in 75 ml tissue cell culture flasks in Eagle's minimal essential medium (EMEM) augmented with 10% fetal bovine serum at 37°C in a humidified atmosphere with 5% CO2. For treatment with fatty acids or analogues, the cells were seeded at a density of 2 × 106 cells in a T25-cm2 flask for 24 h. Each FA or analogue was added to the culture medium as a fatty acid-bovine serum albumin (BSA) complex (2.5:1, FA:BSA) to give the desired final concentration. The controls in these experiments were HepG2 cells with BSA alone. After 24 h treatment, the media was collected, cells were rinsed twice with PBS and cells were harvested for analysis. To each cell suspension prior to lipid extraction a standard mixture of 25 µg C15:0 PE, C17:0 PC and C71:1 TAG was added as standards. Extraction of lipids was performed according to the Folch method. For metabolomics analysis, the lipid extracts were resuspended in chloroform/methanol, 2:1 (v/v).All analyses were carried out using an Agilent 1200 Series HPLC, ACE 5 C8-300 column (2.1 x 100 mm) and linear gradient elution at a flow rate of 0.1 mL/min. Mobile phase A and B consisted of 0.1% formic acid; 10 mM ammonium acetate in H2O and 0.1% formic acid; 10 mM ammonium acetate in ACN/isopropanol (50/50; v/v), respectively. The injection volume was 4 µL. Separation of metabolites was achieved at the following gradient: T=0 min: 30% B; T=1 min: 30% B; T=25 min: 100% B; T=45 min: 100% B; T=47 min: 30% B; and T=60 min: 30% B (re-equilibration). The HPLC system was directly coupled to a Bruker Soalrix 70 Hybrid FTMS instrument equipped with electrospray ionization source (ESI) (Bruker Daltonics). The system was controlled by HyStar v.3.4.8.0 software. MS data was collected with resolving power of 78,000 (at m/z 400) in positive or negative mode under following conditions: a capillary voltage of (+/-) 4,500 V and an end plate offset of -500 V. The dry temperature was set at 180°C. Dry gas flow was maintained 4 L/min. Acquisition range was 244-1,800 m/z with 0.2 s ion accumulation time. LC-MS data was converted into mzXML format using CompassXport v. 3.0.6 and processed by mzMine v.2.10 [25] or XCMS data analysis software. Data processing involved mass detection, chromatographic peak detection and deconvolution, isotopic peaks grouping, normalization and peak alignment. Metabolite data were mean-centered and unit-variance scaled to remove the offsets and adjust the importance of high and low abundance metabolites to an equal level. Significantly altered metabolites were defined by a fold change (FC) >2 and p<0.05. Principal component analysis (PCA) and hierarchical clustering analysis (HCA) of signature metabolites altered in compounds treated cells compared to control were performed in the Metaboanalyst web portal (www.metaboanalyst.ca).
Institute
University of Nebraska-Lincoln
DepartmentBiochemistry
LaboratoryDiRusso Black FATTT Lab
Last NameDiRusso
First NameConcetta
AddressDepartment of Biochemistry, University of Nebraska-Lincoln, N241 Beadle Center 1901 Vine St.
Emailcdirusso2@unl.edu
Phone402-472-6504 or 402-613-9293
Submit Date2015-09-09
Num Groups8
Total Subjects24+24=48
Study Comments8 groups in triplicate ran in both negative and positive mode
Raw Data AvailableYes
Raw Data File Type(s)mzXML
Analysis Type DetailLC-MS
Release Date2017-07-10
Release Version1
Concetta DiRusso Concetta DiRusso
https://dx.doi.org/10.21228/M89W2D
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR000194
Project DOI:doi: 10.21228/M89W2D
Project Title:Cyclobutene- and cyclobutane-functionalized fatty acids as novel biochemical probes of structure and function in HepG2 cells
Project Type:Lipidomics
Project Summary:Five analogues of OA (18:1cis9) or elaidic acid (18:1trans9) replacing the alkene with a four-membered carbocycle were evaluated in HepG2 cells, which are well-characterized models for hepatocytes. In order to assess whether or not the novel analogues were incorporated into complex lipids, cells were treated with compond and then lipids extracted. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) was employed for the analysis of complex lipids. Data processing involved mass detection, chromatographic peak detection and deconvolution, isotopic peaks grouping, normalization and peak alignment. Significantly altered metabolites were defined by a fold change (FC) >2 and p<0.05. Principal component analysis (PCA) and hierarchical clustering analysis (HCA) of signature metabolites altered in compounds treated cells compared to control were performed in the Metaboanalyst web portal (www.metaboanalyst.ca).
Institute:University of Nebraska-Lincoln
Department:Biochemistry
Laboratory:DiRusso Black FATTT Lab
Last Name:DiRusso
First Name:Concetta
Address:Department of Biochemistry, University of Nebraska-Lincoln, N241 Beadle Center 1901 Vine St.
Email:cdirusso2@unl.edu
Phone:402-472-6504 or 402-613-9293
Funding Source:NIH

Subject:

Subject ID:SU000261
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Cell Biosource Or Supplier:ATCC
Cell Strain Details:HepG2
Subject Comments:NA
Cell Primary Immortalized:Immortalized
Cell Passage Number:NA
Cell Counts:NA
Species Group:Human

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Concentration (uM) Treatment_compound
SA011136Control10 BSA
SA011137Control30 BSA
SA011138Control20 BSA
SA011139FAC3500 CB-cis
SA011140FAC2500 CB-cis
SA011141FAC1500 CB-cis
SA011142FAT3500 CB-trans
SA011143FAT2500 CB-trans
SA011144FAT1500 CB-trans
SA011145CKC2500 CK-cis
SA011146CKC3500 CK-cis
SA011147CKC1500 CK-cis
SA011148CKT3500 CK-trans
SA011149CKT2500 CK-trans
SA011150CKT1500 CK-trans
SA011151KC2500 K-cis
SA011152KC3500 K-cis
SA011153KC1500 K-cis
SA011154OA2500 Oleic acid
SA011155OA3500 Oleic acid
SA011156OA1500 Oleic acid
SA011157PA3500 Palmitic acid
SA011158PA1500 Palmitic acid
SA011159PA2500 Palmitic acid
Showing results 1 to 24 of 24

Collection:

Collection ID:CO000252
Collection Summary:-
Sample Type:Cultured cells

Treatment:

Treatment ID:TR000272
Treatment Summary:Cells treated 24hr with 500 µM Fa, analogue or BSA control. Harvested and lipids extracted and then resolved using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) s
Treatment Protocol ID:CBC_treatment
Treatment Protocol Filename:See_Comments
Treatment Protocol Comments:Human hepatoma HepG2 cells (American Type Culture Collection; HB-8065) were grown in 75 ml tissue cell culture flasks in Eagle's minimal essential medium (EMEM) augmented with 10% fetal bovine serum at 37°C in a humidified atmosphere with 5% CO2. For treatment with fatty acids or analogues, the cells were seeded at a density of 2 × 106 cells in a T25-cm2 flask for 24 h. Each FA or analogue was added to the culture medium as a fatty acid-bovine serum albumin (BSA) complex (2.5:1, FA:BSA) to give 500µM final concentration. The controls in these experiments were HepG2 cells with BSA alone. After 24 h treatment, the media was collected, cells were rinsed twice with PBS and cells were harvested for complex lipid analysis.
Treatment Compound:Fatty acid/BSA
Treatment Dose:500 µM (fatty acid)
Treatment Doseduration:24 hr
Treatment Vehicle:PBS
Cell Growth Container:75 ml tissue cell culture flasks
Cell Media:Eagle's minimal essential medium (EMEM) augmented with 10% fetal bovine serum
Cell Envir Cond:37°C in a humidified atmosphere with 5% CO2
Cell Harvesting:Typsinize and scrape
Cell Media Lastchanged:24 hr

Sample Preparation:

Sampleprep ID:SP000266
Sampleprep Summary:-
Sampleprep Protocol Comments:For metabolomics analysis, the lipid extracts were resuspended in chloroform/methanol, 2:1 (v/v).All analyses were carried out using an Agilent 1200 Series HPLC, ACE 5 C8-300 column (2.1 x 100 mm) and linear gradient elution at a flow rate of 0.1 mL/min. Mobile phase A and B consisted of 0.1% formic acid; 10 mM ammonium acetate in H2O and 0.1% formic acid; 10 mM ammonium acetate in ACN/isopropanol (50/50; v/v), respectively. The injection volume was 4 µL. Separation of metabolites was achieved at the following gradient: T=0 min: 30% B; T=1 min: 30% B; T=25 min: 100% B; T=45 min: 100% B; T=47 min: 30% B; and T=60 min: 30% B (re-equilibration). The HPLC system was directly coupled to a Bruker Soalrix 70 Hybrid FTMS instrument equipped with electrospray ionization source (ESI) (Bruker Daltonics). The system was controlled by HyStar v.3.4.8.0 software. MS data was collected with resolving power of 78,000 (at m/z 400) in positive or negative mode under following conditions: a capillary voltage of (+/-) 4,500 V and an end plate offset of -500 V. The dry temperature was set at 180°C. Dry gas flow was maintained 4 L/min. Acquisition range was 244-1,800 m/z with 0.2 s ion accumulation time. LC-MS data was converted into mzXML format using CompassXport v. 3.0.6 and processed by mzMine v.2.10 [25] or XCMS data analysis software. Data processing involved mass detection, chromatographic peak detection and deconvolution, isotopic peaks grouping, normalization and peak alignment.
Extraction Method:Folche Lipid Extraction: Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J biol Chem. 1957;226:497-509.
Sample Spiking:25 µg C15:0 PE, C17:0 PC and C71:1 TAG added to 2 X 10e6 cells prior to lipid extraction
Cell Type:HepG2

Combined analysis:

Analysis ID AN000373 AN000374
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Agilent 1200 Agilent 1200
Column ACE 5 C8-300 (100 x 2.1mm) ACE 5 C8-300 (100 x 2.1mm)
MS Type ESI ESI
MS instrument type FT-ICR-MS FT-ICR-MS
MS instrument name Bruker SolariX FT-ICR-MS Bruker SolariX FT-ICR-MS
Ion Mode POSITIVE NEGATIVE
Units Peak area Peak area

Chromatography:

Chromatography ID:CH000270
Methods Filename:jac8lipidneg.m
Chromatography Comments:jalcms1lipneg.m
Instrument Name:Agilent 1200
Column Name:ACE 5 C8-300 (100 x 2.1mm)
Flow Rate:0.1 mL/min
Internal Standard:25 g C15:0 PE, C17:0 PC and C71:1 TAG
Solvent A:100% water; 0.1% acetic acid; 2 mM ammonium acetate
Solvent B:50% acetonitrile/50% isopropanol; 0.1% formic acid; 10 mM ammonium acetate
Analytical Time:60 min
Chromatography Type:Reversed phase

MS:

MS ID:MS000319
Analysis ID:AN000373
Instrument Name:Bruker SolariX FT-ICR-MS
Instrument Type:FT-ICR-MS
MS Type:ESI
Ion Mode:POSITIVE
Dataformat:*.d
  
MS ID:MS000320
Analysis ID:AN000374
Instrument Name:Bruker SolariX FT-ICR-MS
Instrument Type:FT-ICR-MS
MS Type:ESI
Ion Mode:NEGATIVE
Dataformat:*.d
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