{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002334","ANALYSIS_ID":"AN003811","VERSION":"1","CREATED_ON":"02-08-2024"},

"PROJECT":{"PROJECT_TITLE":"Phospholipase D3 impact on the endolysosomal lipidome","PROJECT_TYPE":"Lipidomics level 2 type quantification as defined by the LSI","PROJECT_SUMMARY":"Lipidomic analysis of isolated endolysosomes phospholipase D3 (PLD3) knockout and rescues with the wild-type, PLD3-M6R and PLD3-V232M sequences.","INSTITUTE":"VIB-KU Leuven","DEPARTMENT":"Center for Brain & Disease Research","LABORATORY":"Laboratory for Membrane Trafficking","LAST_NAME":"Van Acker","FIRST_NAME":"Zoë","ADDRESS":"Herestraat 49 - box 602, 3000 Leuven, Belgium","EMAIL":"zoe.vanacker@kuleuven.vib.be","PHONE":"+32 16 32 07 84","DOI":"http://dx.doi.org/10.21228/M8740F","CONTRIBUTORS":"Lipid analysis was performed at Lipometrix - KU Leuven Lipidomics Core Facility (Leuven, Belgium)."},

"STUDY":{"STUDY_TITLE":"Phospholipase D3 impact on the endolysosomal lipidome","STUDY_SUMMARY":"Neurons rely on the endo-lysosomal network for the maintenance of lipid turnover, removal of dysfunctional organelles and the recycling of proteins. These mechanisms appear to go awry in late-onset Alzheimer’s disease (LOAD). Interestingly, GWA-studies identified risk genes for LOAD linked to endocytic transport regulation (BIN1-CD2AP-PICALM-RIN3-SORL1) and lysosomes (PLD3). Phospholipase D3, also known as PLD3, is a single-pass type II membrane protein that is majorly localized to lysosomes, making it one of the few (or only) risk factors that potentially links lysosomal dysfunction directly to LOAD initiation and progression. CRISPR/Cas9 gene editing was used to generate PLD3 knockout SH-SY5Y cells that were subsequently stably rescued with wild-type PLD3 and coding-variants (M6R & V232M). All cell lines were evaluated for morphological and functional alterations of the endolysosomal compartment, including lipid profiling of endolysosomes magnetically isolated from the different cell lines, as previously described (DOI: 10.1016/j.xpro.2020.100122). A prior isolation step has the unique advantage that it provides spatial resolution to the identified dysregulated networks or compositions. We observe a marked accumulation of storage lipids in endolysosomal isolates; chiefly attributed to cholesterol ester (CE) accretion. A significantly lowered monoacylglycerol level and increased phosphatidylinositol level point to an affected transport/sorting (vesicle/tubule formation).","INSTITUTE":"VIB-KU Leuven","DEPARTMENT":"Center for Brain & Disease Research","LABORATORY":"Laboratory for Membrane Trafficking","LAST_NAME":"Van Acker","FIRST_NAME":"Zoë","ADDRESS":"Herestraat 49 - box 602, 3000 Leuven, Belgium","EMAIL":"zoe.vanacker@kuleuven.vib.be","PHONE":"+3216320784","SUBMIT_DATE":"2022-10-27"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","GENDER":"Female","CELL_STRAIN_DETAILS":"The SH-SY5Y cell line is a thrice cloned subline of the neuroblastoma cell line SK-N-SH (ATCC HTB-11), which was established in 1970 from a metastatic bone tumor from a 4-year-old cancer patient."},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"1",
"Factors":{"Biological replicate":"I"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6","Type":"PNS","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"2",
"Factors":{"Biological replicate":"I"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6","Type":"Bnd/endolysosomal","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"3",
"Factors":{"Biological replicate":"II"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6","Type":"PNS","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"4",
"Factors":{"Biological replicate":"II"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6","Type":"Bnd/endolysosomal","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"5",
"Factors":{"Biological replicate":"III"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6","Type":"PNS","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"6",
"Factors":{"Biological replicate":"III"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6","Type":"Bnd/endolysosomal","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"7",
"Factors":{"Biological replicate":"IV"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x Wt","Type":"PNS","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"8",
"Factors":{"Biological replicate":"IV"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x Wt","Type":"Bnd/endolysosomal","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"17",
"Factors":{"Biological replicate":"IX"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x M6R","Type":"PNS","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"18",
"Factors":{"Biological replicate":"IX"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x M6R","Type":"Bnd/endolysosomal","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"10",
"Factors":{"Biological replicate":"V"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x Wt","Type":"Bnd/endolysosomal","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"9",
"Factors":{"Biological replicate":"V"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x Wt","Type":"PNS","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"11",
"Factors":{"Biological replicate":"VI"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x Wt","Type":"PNS","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"12",
"Factors":{"Biological replicate":"VI"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x Wt","Type":"Bnd/endolysosomal","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"13",
"Factors":{"Biological replicate":"VII"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x M6R","Type":"PNS","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"14",
"Factors":{"Biological replicate":"VII"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x M6R","Type":"Bnd/endolysosomal","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"15",
"Factors":{"Biological replicate":"VIII"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x M6R","Type":"PNS","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"16",
"Factors":{"Biological replicate":"VIII"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x M6R","Type":"Bnd/endolysosomal","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"19",
"Factors":{"Biological replicate":"X"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x V232M","Type":"PNS","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"20",
"Factors":{"Biological replicate":"X"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x V232M","Type":"Bnd/endolysosomal","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"21",
"Factors":{"Biological replicate":"XI"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x V232M","Type":"PNS","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"22",
"Factors":{"Biological replicate":"XI"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x V232M","Type":"Bnd/endolysosomal","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"23",
"Factors":{"Biological replicate":"XII"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x V232M","Type":"PNS","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
},
{
"Subject ID":"-",
"Sample ID":"24",
"Factors":{"Biological replicate":"XII"},
"Additional sample data":{"SH-SY5Y cell line":"PLD3 Knockout ex6 x V232M","Type":"Bnd/endolysosomal","RAW_FILE_NAME":"PLD3.scan PLD3.wiff","Analysed data":"PLD3 lipidomics results (Excel worksheet names represent displayed analyzes and units)"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"High-yield purification of lysosomes was performed according to DOI: 10.1016/j.xpro.2020.100122.","SAMPLE_TYPE":"Cultured cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"No treatment. SH-SY5Y neuroblastoma cells were grown in complete medium: DMEM, high glucose, Glutamax, pyruvate (31966-047) completed with 10% v/v FBS.","CELL_MEDIA":"Complete medium: DMEM, high glucose, Glutamax, pyruvate (31966-047) completed with 10% v/v FBS."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Lipid analysis was performed at Lipometrix - KU Leuven Lipidomics Core Facility (Leuven, Belgium). An amount of sample containing 10 ug of protein was diluted in 700 μl water and mixed with 800 μl 1 N HCl:CH3OH 1:8 (v/v), 900 μl CHCl3, 200 μg/ml of the antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT; Sigma Aldrich) and 3 μl of SPLASH® LIPIDOMIX® Mass Spec Standard (#330707, Avanti Polar Lipids). After vortexing and centrifugation, the lower organic fraction was collected and evaporated using a Savant Speedvac spd111v (Thermo Fisher Scientific) at room temperature and the remaining lipid pellet was stored at - 20°C under argon. Just before mass spectrometry analysis, lipid pellets were reconstituted in 100% ethanol. Lipid species were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) on a Nexera X2 UHPLC system (Shimadzu) coupled with hybrid triple quadrupole/linear ion trap mass spectrometer (6500+ QTRAP system; AB SCIEX). Chromatographic separation was performed on a XBridge amide column (150 mm × 4.6 mm, 3.5 μm; Waters) maintained at 35°C using mobile phase A [1 mM ammonium acetate in water-acetonitrile 5:95 (v/v)] and mobile phase B [1 mM ammonium acetate in water-acetonitrile 50:50 (v/v)] in the following gradient: (0-6 min: 0% B  6% B; 6-10 min: 6% B  25% B; 10-11 min: 25% B  98% B; 11-13 min: 98% B  100% B; 13-19 min: 100% B; 19-24 min: 0% B) at a flow rate of 0.7 mL/min, which was increased to 1.5 mL/min from 13 minutes onwards. SM, CE, CER, DCER, HCER, LCER were measured in positive ion mode with a precursor scan of 184.1, 369.4, 264.4, 266.4, 264.4 and 264.4 respectively. TAG, DAG and MAG were measured in positive ion mode with a neutral loss scan for one of the fatty acyl moieties. PC, LPC, PE, LPE, PG, PI and PS were measured in negative ion mode by fatty acyl fragment ions. Lipid quantification was performed by scheduled multiple reactions monitoring (MRM), the transitions being based on the neutral losses or the typical product ions as described above. The instrument parameters were as follows: Curtain Gas = 35 psi; Collision Gas = 8 a.u. (medium); IonSpray Voltage = 5500 V and −4,500 V; Temperature = 550°C; Ion Source Gas 1 = 50 psi; Ion Source Gas 2 = 60 psi; Declustering Potential = 60 V and −80 V; Entrance Potential = 10 V and −10 V; Collision Cell Exit Potential = 15 V and −15 V. The following fatty acyl moieties were taken into account for the lipidomic analysis: 14:0, 14:1, 16:0, 16:1, 16:2, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 20:4, 20:5, 22:0, 22:1, 22:2, 22:4, 22:5 and 22:6 except for TGs which considered: 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:3, 20:4, 20:5, 22:2, 22:3, 22:4, 22:5, 22:6. Peak integration was performed with the MultiQuantTM software version 3.0.3. Lipid species signals were corrected for isotopic contributions (calculated with Python Molmass 2019.1.1) and were quantified based on internal standard signals and adheres to the guidelines of the Lipidomics Standards Initiative (LSI) (level 2 type quantification as defined by the LSI)."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Chromatographic separation was performed on a XBridge amide column (150 mm × 4.6 mm, 3.5 μm; Waters) maintained at 35°C using mobile phase A [1 mM ammonium acetate in water-acetonitrile 5:95 (v/v)] and mobile phase B [1 mM ammonium acetate in water-acetonitrile 50:50 (v/v)] in the following gradient: (0-6 min: 0% B - 6% B; 6-10 min: 6% B - 25% B; 10-11 min: 25% B - 98% B; 11-13 min: 98% B - 100% B; 13-19 min: 100% B; 19-24 min: 0% B) at a flow rate of 0.7 mL/min, which was increased to 1.5 mL/min from 13 minutes onwards.","INSTRUMENT_NAME":"Shimadzu Nexera X2","COLUMN_NAME":"Waters XBridge Amide (100 x 4.6mm,3.5um)","COLUMN_TEMPERATURE":"35","FLOW_GRADIENT":"0-6 min: 0% B - 6% B; 6-10 min: 6% B - 25% B; 10-11 min: 25% B - 98% B; 11-13 min: 98% B - 100% B; 13-19 min: 100% B; 19-24 min: 0% B","FLOW_RATE":"0.7 mL/min, which was increased to 1.5 mL/min from 13 minutes onwards","SOLVENT_A":"5% water/95% acetonitrile; 1 mM ammonium acetate","SOLVENT_B":"50% water/50% acetonitrile; 1 mM ammonium acetate","CHROMATOGRAPHY_TYPE":"HILIC"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"ABI Sciex 6500 QTrap","INSTRUMENT_TYPE":"hybrid triple quadrupole/linear ion trap","MS_TYPE":"ESI","MS_COMMENTS":"PC, LPC, PE, LPE, PG, PI and PS were measured in negative ion mode by fatty acyl fragment ions. Lipid quantification was performed by scheduled multiple reactions monitoring (MRM), the transitions being based on the neutral losses or the typical product ions as described above. The instrument parameters were as follows: Curtain Gas = 35 psi; Collision Gas = 8 a.u. (medium); IonSpray Voltage = 5500 V and −4,500 V; Temperature = 550°C; Ion Source Gas 1 = 50 psi; Ion Source Gas 2 = 60 psi; Declustering Potential = 60 V and −80 V; Entrance Potential = 10 V and −10 V; Collision Cell Exit Potential = 15 V and −15 V.","ION_MODE":"NEGATIVE"},

"MS_METABOLITE_DATA":{
"Units":"nmol lipid per mg protein",

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