Summary of Study ST002185

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 PR001392. The data can be accessed directly via it's Project DOI: 10.21228/M8SH87 This work is supported by NIH grant, U2C- DK119886.

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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 IDST002185
Study TitleLipidomic characterization of Jurkat-derived T cell line in which the chaperonin complex CCT has been partially silenced
Study TypeUntargeted Lipidomics
Study SummaryWhen the chaperonin complex CCT is partially silenced in Jurkat-derived T cell line J77 E61, we observe changes in the production of exosomes and in their composition. Thus, to explore the bases of these alterations and find possible new mechanisms of exosome biosynthesis regulation we characterized the lipidome content in cells where the chaperonin complex CCT was partially silent (CCT) and controls (CTRL). We analyzed 5 biological replicates containing 3 × 106 cells using LC-MS in positive and negative polarity mode. For quality control, 5 QCs samples and 4 blanks were also included in the analysis (total 19 samples and 2 groups).
Institute
Centro Nacional de Investigaciones Cardiovasculares Carlos III
DepartmentProteomics and Metabolomics Unit
LaboratoryMetabolomics Lab
Last NameFerrarini
First NameAlessia
AddressCalle de Melchor Fernández Almagro, 3, Madrid, Madrid, 28029, Spain
Emailaferrarini@cnic.es
Phone914 53 12 00
Submit Date2022-06-03
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2023-06-06
Release Version1
Alessia Ferrarini Alessia Ferrarini
https://dx.doi.org/10.21228/M8SH87
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001392
Project DOI:doi: 10.21228/M8SH87
Project Title:Chaperonin CCT controls cell fueling by lipids and extracellular vesicle production through kinesin dynamics
Project Type:Untargeted Lipidomics
Project Summary:Cells regulate their protein content through different processes including gene transcription, protein translation, post-translational modification, secretion, degradation and recycling. The complexity of this network and its dynamic regulation remains mostly unexplored. A plethora of intracellular elements can be incorporated into multivesicular bodies (MVB) to be secreted as soluble components or extracellular vesicles (EVs). By profiling the proteome of EVs from T cells, we have found the subunits of the chaperonin CCT, involved in the correct folding of particular proteins. By limiting CCT content after siRNA silencing, cells shift the dynamics of lipid droplets, peroxisomes and the endolysosomal system, showing an accumulation of MVBs that leads to increased EV production and an altered lipid composition. Also, their metabolic profile is shifted towards a lipid-dependent metabolism. This is exerted through the dynamic regulation of microtubule-based kinesin motor.
Institute:Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC)
Department:Proteomics and Metabolomics Unit
Laboratory:Metabolomics Lab
Last Name:Ferrarini
First Name:Alessia
Address:Calle de Melchor Fernández Almagro, 3, Madrid, Madrid, 28029, Spain
Email:aferrarini@cnic.es
Phone:914 53 12 00

Subject:

Subject ID:SU002271
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Genotype Strain:E6-1
Gender:Not applicable
Cell Biosource Or Supplier:human blood (leukemic T-cell lymphoblast) - from Dr. A. Weiss, NIH AIDS Reagent Program, Division of 554 AIDS, NIAID, NIH
Cell Counts:3 x 10e6

Factors:

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

mb_sample_id local_sample_id Treatment Replicate
SA210010N_BlankA_02Extraction Blank -
SA210011N_BlankA_01Extraction Blank -
SA210012P_BlankA_01Extraction Blank 1
SA210013P_BlankA_02Extraction Blank 2
SA210014N_QC_01QC -
SA210015N_QC_02QC -
SA210016N_QC_4QC -
SA210017N_QC_5QC -
SA210018N_QC_3QC -
SA210019P_QC_01QC 1
SA210020P_QC_02QC 2
SA210021P_QC_3QC 3
SA210022P_QC_4QC 4
SA210023P_QC_5QC 5
SA210028N_CCT_02siCCT -
SA210029N_CCT_03siCCT -
SA210030N_CCT_04siCCT -
SA210031N_CCT_01siCCT -
SA210032N_CCT_05siCCT -
SA210033P_CCT_01siCCT 1
SA210034P_CCT_02siCCT 2
SA210035P_CCT_03siCCT 3
SA210036P_CCT_04siCCT 4
SA210037P_CCT_05siCCT 5
SA210038N_CTRL_05siCTRL -
SA210039N_CTRL_04siCTRL -
SA210040N_CTRL_02siCTRL -
SA210041N_CTRL_01siCTRL -
SA210042N_CTRL_03siCTRL -
SA210043P_CTRL_01siCTRL 1
SA210044P_CTRL_02siCTRL 2
SA210045P_CTRL_03siCTRL 3
SA210046P_CTRL_04siCTRL 4
SA210047P_CTRL_05siCTRL 5
SA210024N_Blank_2Solvent Blank -
SA210025N_Blank_1Solvent Blank -
SA210026P_Blank_1Solvent Blank 1
SA210027P_Blank_2Solvent Blank 2
Showing results 1 to 38 of 38

Collection:

Collection ID:CO002264
Collection Summary:The Jurkat E6-1 cell line was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. A. Weiss and grown in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Invitrogen).
Sample Type:T-cells
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002283
Treatment Summary:E6-1 Jurkat cells were transfected with a pool of double-stranded siRNAs targeting CCT1 (5’-CCAUUGGAGACUUGGUAAA-3’), CCT2 (5’-UGGUAAACCUCGAGACAAC-3’), CCT4 (5’- AGGUGGUGCUCCAGAAAUA-3’) and CCT5 (5’-CCAGACAGGUGAAGGAGAU-3’). As control, cells were transfected with a nonspecific siRNA (5’-UUCUCCGAACGUGUGCACG-3’). Cells were resuspended in Opti-MEM (Gibco; 1 x 106 cells per ml) with 1.2 M of each siRNA to a total of 5 M and electroporated with Gene PulserXcell (Bio-Rad) at 240 mV and 95 mA in 4 mm cuvettes (Bio-Rad). Silencing was effective from 48 h until 72 h after the transfection step.

Sample Preparation:

Sampleprep ID:SP002277
Sampleprep Summary:Five biological replicate containing 3 × 106 cells were collected and stored at -80°C. The cell pellets were thaw on ice and subjected to three freeze–thaw cycles for complete cell disruption and protein precipitation. Briefly, samples were suspended in 250 µL freshly prepared methanol:acetic acid (98:2 v/v) solution, vortex-mixed, placed in liquid nitrogen for 10s and thaw in an ice bath (for 10s) three times. Subsequently samples were centrifuge at 18000g for 20 min. at 10°C. Supernatants were collected and lipids were extracted with methyl-tert-butylether (MTBE) as described (Matyash et al., 2008). 400 µL of organic phase were dried-out in speedvac and resuspended in 50 µL of ACN:H2O (20:80, v:v) just before injection. In order to assess the reproducibility and robusteness of the methodology, quality control samples (QC) were prepared by polling together one CCT and one CTRL sample’s supernatant and following the same extraction procedures. Dried extracted were resuspended in 50 µL of ACN:H2O (20:80, v:v) just before injection.
Processing Storage Conditions:Described in summary
Extract Storage:Described in summary

Combined analysis:

Analysis ID AN003578 AN003579
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Dionex Ultimate 3000 Thermo Dionex Ultimate 3000
Column mRP-Recovery C18 (100 × 0.5 mm,5um) mRP-Recovery C18 (100 × 0.5 mm,5um)
MS Type ESI ESI
MS instrument type LTQ-FT LTQ-FT
MS instrument name Thermo Orbitrap Elite Hybrid Ion Trap-Orbitrap Thermo Orbitrap Elite Hybrid Ion Trap-Orbitrap
Ion Mode POSITIVE NEGATIVE
Units log Abundance log Abundance

Chromatography:

Chromatography ID:CH002646
Chromatography Summary:Lipidomics untargeted analysis was performed using an Ultimate 3000 HPLC equipped with Agilent mRP-Recovery C18 column (100 × 0.5 mm, 5 µm) thermostated at 55°C. Lipids were eluted at 100 mL/min using (A) water + 0.1% of formic acid and (B) acetonitrile with 0.1% formic acid. MS was operating in full scan mode from 70 to 1700 m/z at 60000 resolution in positive and negative polarity mode, in separate runs.
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:mRP-Recovery C18 (100 × 0.5 mm,5um)
Flow Rate:100 mL/min
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS003335
Analysis ID:AN003578
Instrument Name:Thermo Orbitrap Elite Hybrid Ion Trap-Orbitrap
Instrument Type:LTQ-FT
MS Type:ESI
MS Comments:Lipidomics untargeted analysis was performed using an Ultimate 3000 HPLC equipped with Agilent mRP-Recovery C18 column (100 × 0.5 mm, 5 µm) thermostated at 55°C, coupled to an Orbitrap ELITE™ Hybrid Ion Trap-Orbitrap Mass Spectrometer (ThermoFisher Scientific). MS was operating in full scan mode from 70 to 1700 m/z at 60000 resolution in positive and negative polarity mode, in separate runs. Data processing was carried-out using Compound Discoverer (ThermoFisher; USA) with the Metaboprofiler node (Röst et al., 2016) and MetaboAnalyst (Pang et al., 2021).
Ion Mode:POSITIVE
  
MS ID:MS003336
Analysis ID:AN003579
Instrument Name:Thermo Orbitrap Elite Hybrid Ion Trap-Orbitrap
Instrument Type:LTQ-FT
MS Type:ESI
MS Comments:Lipidomics untargeted analysis was performed using an Ultimate 3000 HPLC equipped with Agilent mRP-Recovery C18 column (100 × 0.5 mm, 5 µm) thermostated at 55°C, coupled to an Orbitrap ELITE™ Hybrid Ion Trap-Orbitrap Mass Spectrometer (ThermoFisher Scientific). MS was operating in full scan mode from 70 to 1700 m/z at 60000 resolution in positive and negative polarity mode, in separate runs. Data processing was carried-out using Compound Discoverer (ThermoFisher; USA) with the Metaboprofiler node (Röst et al., 2016) and MetaboAnalyst (Pang et al., 2021).
Ion Mode:NEGATIVE
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