#METABOLOMICS WORKBENCH aferrarini_20220603_062700 DATATRACK_ID:3292 STUDY_ID:ST002185 ANALYSIS_ID:AN003579 PROJECT_ID:PR001392
VERSION             	1
CREATED_ON             	June 3, 2022, 7:57 am
#PROJECT
PR:PROJECT_TITLE                 	Chaperonin CCT controls cell fueling by lipids and extracellular vesicle
PR:PROJECT_TITLE                 	production through kinesin dynamics
PR:PROJECT_TYPE                  	Untargeted Lipidomics
PR:PROJECT_SUMMARY               	Cells regulate their protein content through different processes including gene
PR:PROJECT_SUMMARY               	transcription, protein translation, post-translational modification, secretion,
PR:PROJECT_SUMMARY               	degradation and recycling. The complexity of this network and its dynamic
PR:PROJECT_SUMMARY               	regulation remains mostly unexplored. A plethora of intracellular elements can
PR:PROJECT_SUMMARY               	be incorporated into multivesicular bodies (MVB) to be secreted as soluble
PR:PROJECT_SUMMARY               	components or extracellular vesicles (EVs). By profiling the proteome of EVs
PR:PROJECT_SUMMARY               	from T cells, we have found the subunits of the chaperonin CCT, involved in the
PR:PROJECT_SUMMARY               	correct folding of particular proteins. By limiting CCT content after siRNA
PR:PROJECT_SUMMARY               	silencing, cells shift the dynamics of lipid droplets, peroxisomes and the
PR:PROJECT_SUMMARY               	endolysosomal system, showing an accumulation of MVBs that leads to increased EV
PR:PROJECT_SUMMARY               	production and an altered lipid composition. Also, their metabolic profile is
PR:PROJECT_SUMMARY               	shifted towards a lipid-dependent metabolism. This is exerted through the
PR:PROJECT_SUMMARY               	dynamic regulation of microtubule-based kinesin motor.
PR:INSTITUTE                     	Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC)
PR:DEPARTMENT                    	Proteomics and Metabolomics Unit
PR:LABORATORY                    	Metabolomics Lab
PR:LAST_NAME                     	Ferrarini
PR:FIRST_NAME                    	Alessia
PR:ADDRESS                       	Calle de Melchor Fernández Almagro, 3, Madrid, Madrid, 28029, Spain
PR:EMAIL                         	aferrarini@cnic.es
PR:PHONE                         	914 53 12 00
#STUDY
ST:STUDY_TITLE                   	Lipidomic characterization of Jurkat-derived T cell line in which the chaperonin
ST:STUDY_TITLE                   	complex CCT has been partially silenced
ST:STUDY_TYPE                    	Untargeted Lipidomics
ST:STUDY_SUMMARY                 	When the chaperonin complex CCT is partially silenced in Jurkat-derived T cell
ST:STUDY_SUMMARY                 	line J77 E61, we observe changes in the production of exosomes and in their
ST:STUDY_SUMMARY                 	composition. Thus, to explore the bases of these alterations and find possible
ST:STUDY_SUMMARY                 	new mechanisms of exosome biosynthesis regulation we characterized the lipidome
ST:STUDY_SUMMARY                 	content in cells where the chaperonin complex CCT was partially silent (CCT) and
ST:STUDY_SUMMARY                 	controls (CTRL). We analyzed 5 biological replicates containing 3 × 106 cells
ST:STUDY_SUMMARY                 	using LC-MS in positive and negative polarity mode. For quality control, 5 QCs
ST:STUDY_SUMMARY                 	samples and 4 blanks were also included in the analysis (total 19 samples and 2
ST:STUDY_SUMMARY                 	groups).
ST:INSTITUTE                     	Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC)
ST:DEPARTMENT                    	Proteomics and Metabolomics Unit
ST:LABORATORY                    	Metabolomics Lab
ST:LAST_NAME                     	Ferrarini
ST:FIRST_NAME                    	Alessia
ST:ADDRESS                       	Calle de Melchor Fernández Almagro, 3, Madrid, Madrid, 28029, Spain
ST:EMAIL                         	aferrarini@cnic.es
ST:PHONE                         	914 53 12 00
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:GENOTYPE_STRAIN               	E6-1
SU:GENDER                        	Not applicable
SU:CELL_BIOSOURCE_OR_SUPPLIER    	human blood (leukemic T-cell lymphoblast) - from Dr. A. Weiss, NIH AIDS Reagent
SU:CELL_BIOSOURCE_OR_SUPPLIER    	Program, Division of 554 AIDS, NIAID, NIH
SU:CELL_COUNTS                   	3 x 10e6
#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           	Solvent Blank	P_Blank_1	Treatment:Solvent Blank | Replicate:1	RAW_FILE_NAME=P_Blank_1.mzML
SUBJECT_SAMPLE_FACTORS           	Solvent Blank	P_Blank_2	Treatment:Solvent Blank | Replicate:2	RAW_FILE_NAME=P_Blank_2.mzML
SUBJECT_SAMPLE_FACTORS           	Extraction Blank	P_BlankA_01	Treatment:Extraction Blank | Replicate:1	RAW_FILE_NAME=P_BlankA_01.mzML
SUBJECT_SAMPLE_FACTORS           	Extraction Blank	P_BlankA_02	Treatment:Extraction Blank | Replicate:2	RAW_FILE_NAME=P_BlankA_02.mzML
SUBJECT_SAMPLE_FACTORS           	CCT	P_CCT_01	Treatment:siCCT | Replicate:1	RAW_FILE_NAME=P_CCT_01.mzML
SUBJECT_SAMPLE_FACTORS           	CCT	P_CCT_02	Treatment:siCCT | Replicate:2	RAW_FILE_NAME=P_CCT_02.mzML
SUBJECT_SAMPLE_FACTORS           	CCT	P_CCT_03	Treatment:siCCT | Replicate:3	RAW_FILE_NAME=P_CCT_03.mzML
SUBJECT_SAMPLE_FACTORS           	CCT	P_CCT_04	Treatment:siCCT | Replicate:4	RAW_FILE_NAME=P_CCT_04.mzML
SUBJECT_SAMPLE_FACTORS           	CCT	P_CCT_05	Treatment:siCCT | Replicate:5	RAW_FILE_NAME=P_CCT_05.mzML
SUBJECT_SAMPLE_FACTORS           	CTRL	P_CTRL_01	Treatment:siCTRL | Replicate:1	RAW_FILE_NAME=P_CTRL_01.mzML
SUBJECT_SAMPLE_FACTORS           	CTRL	P_CTRL_02	Treatment:siCTRL | Replicate:2	RAW_FILE_NAME=P_CTRL_02.mzML
SUBJECT_SAMPLE_FACTORS           	CTRL	P_CTRL_03	Treatment:siCTRL | Replicate:3	RAW_FILE_NAME=P_CTRL_03.mzML
SUBJECT_SAMPLE_FACTORS           	CTRL	P_CTRL_04	Treatment:siCTRL | Replicate:4	RAW_FILE_NAME=P_CTRL_04.mzML
SUBJECT_SAMPLE_FACTORS           	CTRL	P_CTRL_05	Treatment:siCTRL | Replicate:5	RAW_FILE_NAME=P_CTRL_05.mzML
SUBJECT_SAMPLE_FACTORS           	QC	P_QC_01	Treatment:QC | Replicate:1	RAW_FILE_NAME=P_QC_01.mzML
SUBJECT_SAMPLE_FACTORS           	QC	P_QC_02	Treatment:QC | Replicate:2	RAW_FILE_NAME=P_QC_02.mzML
SUBJECT_SAMPLE_FACTORS           	QC	P_QC_3	Treatment:QC | Replicate:3	RAW_FILE_NAME=P_QC_3.mzML
SUBJECT_SAMPLE_FACTORS           	QC	P_QC_4	Treatment:QC | Replicate:4	RAW_FILE_NAME=P_QC_4.mzML
SUBJECT_SAMPLE_FACTORS           	QC	P_QC_5	Treatment:QC | Replicate:5	RAW_FILE_NAME=P_QC_5.mzML
SUBJECT_SAMPLE_FACTORS           	Solvent Blank	N_Blank_1	Treatment:Solvent Blank | Replicate:-	RAW_FILE_NAME=N_Blank_1.mzML
SUBJECT_SAMPLE_FACTORS           	Solvent Blank	N_Blank_2	Treatment:Solvent Blank | Replicate:-	RAW_FILE_NAME=N_Blank_2.mzML
SUBJECT_SAMPLE_FACTORS           	Extraction Blank	N_BlankA_01	Treatment:Extraction Blank | Replicate:-	RAW_FILE_NAME=N_BlankA_01.mzML
SUBJECT_SAMPLE_FACTORS           	Extraction Blank	N_BlankA_02	Treatment:Extraction Blank | Replicate:-	RAW_FILE_NAME=N_BlankA_02.mzML
SUBJECT_SAMPLE_FACTORS           	CCT	N_CCT_01	Treatment:siCCT | Replicate:-	RAW_FILE_NAME=N_CCT_01.mzML
SUBJECT_SAMPLE_FACTORS           	CCT	N_CCT_02	Treatment:siCCT | Replicate:-	RAW_FILE_NAME=N_CCT_02.mzML
SUBJECT_SAMPLE_FACTORS           	CCT	N_CCT_03	Treatment:siCCT | Replicate:-	RAW_FILE_NAME=N_CCT_03.mzML
SUBJECT_SAMPLE_FACTORS           	CCT	N_CCT_04	Treatment:siCCT | Replicate:-	RAW_FILE_NAME=N_CCT_04.mzML
SUBJECT_SAMPLE_FACTORS           	CCT	N_CCT_05	Treatment:siCCT | Replicate:-	RAW_FILE_NAME=N_CCT_05.mzML
SUBJECT_SAMPLE_FACTORS           	CTRL	N_CTRL_01	Treatment:siCTRL | Replicate:-	RAW_FILE_NAME=N_CTRL_01.mzML
SUBJECT_SAMPLE_FACTORS           	CTRL	N_CTRL_02	Treatment:siCTRL | Replicate:-	RAW_FILE_NAME=N_CTRL_02.mzML
SUBJECT_SAMPLE_FACTORS           	CTRL	N_CTRL_03	Treatment:siCTRL | Replicate:-	RAW_FILE_NAME=N_CTRL_03.mzML
SUBJECT_SAMPLE_FACTORS           	CTRL	N_CTRL_04	Treatment:siCTRL | Replicate:-	RAW_FILE_NAME=N_CTRL_04.mzML
SUBJECT_SAMPLE_FACTORS           	CTRL	N_CTRL_05	Treatment:siCTRL | Replicate:-	RAW_FILE_NAME=N_CTRL_05.mzML
SUBJECT_SAMPLE_FACTORS           	QC	N_QC_01	Treatment:QC | Replicate:-	RAW_FILE_NAME=N_QC_01.mzML
SUBJECT_SAMPLE_FACTORS           	QC	N_QC_02	Treatment:QC | Replicate:-	RAW_FILE_NAME=N_QC_02.mzML
SUBJECT_SAMPLE_FACTORS           	QC	N_QC_3	Treatment:QC | Replicate:-	RAW_FILE_NAME=N_QC_3.mzML
SUBJECT_SAMPLE_FACTORS           	QC	N_QC_4	Treatment:QC | Replicate:-	RAW_FILE_NAME=N_QC_4.mzML
SUBJECT_SAMPLE_FACTORS           	QC	N_QC_5	Treatment:QC | Replicate:-	RAW_FILE_NAME=N_QC_5.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	The Jurkat E6-1 cell line was obtained through the NIH AIDS Reagent Program,
CO:COLLECTION_SUMMARY            	Division of AIDS, NIAID, NIH from Dr. A. Weiss and grown in RPMI 1640 medium
CO:COLLECTION_SUMMARY            	(Gibco) supplemented with 10% fetal bovine serum (FBS, Invitrogen).
CO:SAMPLE_TYPE                   	T-cells
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	E6-1 Jurkat cells were transfected with a pool of double-stranded siRNAs
TR:TREATMENT_SUMMARY             	targeting CCT1 (5’-CCAUUGGAGACUUGGUAAA-3’), CCT2
TR:TREATMENT_SUMMARY             	(5’-UGGUAAACCUCGAGACAAC-3’), CCT4 (5’- AGGUGGUGCUCCAGAAAUA-3’) and CCT5
TR:TREATMENT_SUMMARY             	(5’-CCAGACAGGUGAAGGAGAU-3’). As control, cells were transfected with a
TR:TREATMENT_SUMMARY             	nonspecific siRNA (5’-UUCUCCGAACGUGUGCACG-3’). Cells were resuspended in
TR:TREATMENT_SUMMARY             	Opti-MEM (Gibco; 1 x 106 cells per ml) with 1.2 M of each siRNA to a total of
TR:TREATMENT_SUMMARY             	5 M and electroporated with Gene PulserXcell (Bio-Rad) at 240 mV and 95 mA in
TR:TREATMENT_SUMMARY             	4 mm cuvettes (Bio-Rad). Silencing was effective from 48 h until 72 h after the
TR:TREATMENT_SUMMARY             	transfection step.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Five biological replicate containing 3 × 106 cells were collected and stored at
SP:SAMPLEPREP_SUMMARY            	-80°C. The cell pellets were thaw on ice and subjected to three freeze–thaw
SP:SAMPLEPREP_SUMMARY            	cycles for complete cell disruption and protein precipitation. Briefly, samples
SP:SAMPLEPREP_SUMMARY            	were suspended in 250 µL freshly prepared methanol:acetic acid (98:2 v/v)
SP:SAMPLEPREP_SUMMARY            	solution, vortex-mixed, placed in liquid nitrogen for 10s and thaw in an ice
SP:SAMPLEPREP_SUMMARY            	bath (for 10s) three times. Subsequently samples were centrifuge at 18000g for
SP:SAMPLEPREP_SUMMARY            	20 min. at 10°C. Supernatants were collected and lipids were extracted with
SP:SAMPLEPREP_SUMMARY            	methyl-tert-butylether (MTBE) as described (Matyash et al., 2008). 400 µL of
SP:SAMPLEPREP_SUMMARY            	organic phase were dried-out in speedvac and resuspended in 50 µL of ACN:H2O
SP:SAMPLEPREP_SUMMARY            	(20:80, v:v) just before injection. In order to assess the reproducibility and
SP:SAMPLEPREP_SUMMARY            	robusteness of the methodology, quality control samples (QC) were prepared by
SP:SAMPLEPREP_SUMMARY            	polling together one CCT and one CTRL sample’s supernatant and following the
SP:SAMPLEPREP_SUMMARY            	same extraction procedures. Dried extracted were resuspended in 50 µL of
SP:SAMPLEPREP_SUMMARY            	ACN:H2O (20:80, v:v) just before injection.
SP:PROCESSING_STORAGE_CONDITIONS 	Described in summary
SP:EXTRACT_STORAGE               	Described in summary
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Lipidomics untargeted analysis was performed using an Ultimate 3000 HPLC
CH:CHROMATOGRAPHY_SUMMARY        	equipped with Agilent mRP-Recovery C18 column (100 × 0.5 mm, 5 µm)
CH:CHROMATOGRAPHY_SUMMARY        	thermostated at 55°C. Lipids were eluted at 100 mL/min using (A) water + 0.1%
CH:CHROMATOGRAPHY_SUMMARY        	of formic acid and (B) acetonitrile with 0.1% formic acid. MS was operating in
CH:CHROMATOGRAPHY_SUMMARY        	full scan mode from 70 to 1700 m/z at 60000 resolution in positive and negative
CH:CHROMATOGRAPHY_SUMMARY        	polarity mode, in separate runs.
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Dionex Ultimate 3000
CH:COLUMN_NAME                   	mRP-Recovery C18 column (100 × 0.5 mm, 5 µm)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Orbitrap Elite Hybrid Ion Trap-Orbitrap
MS:INSTRUMENT_TYPE               	LTQ-FT
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Lipidomics untargeted analysis was performed using an Ultimate 3000 HPLC
MS:MS_COMMENTS                   	equipped with Agilent mRP-Recovery C18 column (100 × 0.5 mm, 5 µm)
MS:MS_COMMENTS                   	thermostated at 55°C, coupled to an Orbitrap ELITE™ Hybrid Ion Trap-Orbitrap
MS:MS_COMMENTS                   	Mass Spectrometer (ThermoFisher Scientific). MS was operating in full scan mode
MS:MS_COMMENTS                   	from 70 to 1700 m/z at 60000 resolution in positive and negative polarity mode,
MS:MS_COMMENTS                   	in separate runs. Data processing was carried-out using Compound Discoverer
MS:MS_COMMENTS                   	(ThermoFisher; USA) with the Metaboprofiler node (Röst et al., 2016) and
MS:MS_COMMENTS                   	MetaboAnalyst (Pang et al., 2021).
MS:MS_RESULTS_FILE               	ST002185_AN003579_Results.txt	UNITS:log Abundance	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END