#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