{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001673","ANALYSIS_ID":"AN002731","VERSION":"1","CREATED_ON":"February 2, 2021, 10:15 am"},

"PROJECT":{"PROJECT_TITLE":"Targeted Sphingolipid analysis of HeLa silenced for or overexpressing GOLPH3 or LCS","PROJECT_SUMMARY":"A group of sequentially-acting enzymes operating at the branchpoint among sphingolipid synthetic pathways binds the Golgi-localised oncoprotein GOLPH3. GOLPH3 sorts these enzymes into vesicles for intra-Golgi retro-transport, acting as a component of the cisternae inter-conversion mechanisms. Through these effects, GOLPH3 controls the sub-Golgi localisation, and the lysosomal degradation rate of specific enzymes. Here we evaluated the impact of overexpressing or silencing GOLPH3 or its client enzyme lactosylceramide synthase (LCS) on the sphingolipid composition of HeLa cells by targeted lipid analysis.","INSTITUTE":"École polytechnique fédérale de Lausanne (EPFL)","DEPARTMENT":"IBI","LABORATORY":"UPDANGELO","LAST_NAME":"D'Angelo","FIRST_NAME":"Giovanni","ADDRESS":"Station 15, Lausanne, Vaud, 1015, Switzerland","EMAIL":"giovanni.dangelo@epfl.ch","PHONE":"+41 216934276"},

"STUDY":{"STUDY_TITLE":"Targeted Sphingolipid analysis of HeLa silenced for or overexpressing GOLPH3 or LCS","STUDY_SUMMARY":"A group of sequentially-acting enzymes operating at the branchpoint among sphingolipid synthetic pathways binds the Golgi-localised oncoprotein GOLPH3. GOLPH3 sorts these enzymes into vesicles for intra-Golgi retro-transport, acting as a component of the cisternae inter-conversion mechanisms. Through these effects, GOLPH3 controls the sub-Golgi localisation, and the lysosomal degradation rate of specific enzymes. Here we evaluated the impact of overexpressing or silencing GOLPH3 or its client enzyme lactosylceramide synthase (LCS) on the sphingolipid composition of HeLa cells by targeted lipid analysis.","INSTITUTE":"École polytechnique fédérale de Lausanne (EPFL)","DEPARTMENT":"IBI","LABORATORY":"UPDANGELO","LAST_NAME":"D'Angelo","FIRST_NAME":"Giovanni","ADDRESS":"Station 15, Lausanne, Vaud, 1015, Switzerland","EMAIL":"giovanni.dangelo@epfl.ch","PHONE":"+41 216934276"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"CTRL_1",
"Factors":{"Treatment":"no treatment"},
"Additional sample data":{"RAW_FILE_NAME":"CTRL_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"CTRL_2",
"Factors":{"Treatment":"no treatment"},
"Additional sample data":{"RAW_FILE_NAME":"CTRL_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"CTRL_3",
"Factors":{"Treatment":"no treatment"},
"Additional sample data":{"RAW_FILE_NAME":"CTRL_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"GOLPH3_OE_1",
"Factors":{"Treatment":"GOLPH3 overexpression"},
"Additional sample data":{"RAW_FILE_NAME":"GOLPH3_OE_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"GOLPH3_OE_2",
"Factors":{"Treatment":"GOLPH3 overexpression"},
"Additional sample data":{"RAW_FILE_NAME":"GOLPH3_OE_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"GOLPH3_OE_3",
"Factors":{"Treatment":"GOLPH3 overexpression"},
"Additional sample data":{"RAW_FILE_NAME":"GOLPH3_OE_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"GOLPH3_KD_1",
"Factors":{"Treatment":"GOLPH3 silencing"},
"Additional sample data":{"RAW_FILE_NAME":"GOLPH3_KD_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"GOLPH3_KD_2",
"Factors":{"Treatment":"GOLPH3 silencing"},
"Additional sample data":{"RAW_FILE_NAME":"GOLPH3_KD_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"GOLPH3_KD_3",
"Factors":{"Treatment":"GOLPH3 silencing"},
"Additional sample data":{"RAW_FILE_NAME":"GOLPH3_KD_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"LCS_OE_1",
"Factors":{"Treatment":"LCS overexpression"},
"Additional sample data":{"RAW_FILE_NAME":"LCS_OE_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"LCS_OE_2",
"Factors":{"Treatment":"LCS overexpression"},
"Additional sample data":{"RAW_FILE_NAME":"LCS_OE_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"LCS_OE_3",
"Factors":{"Treatment":"LCS overexpression"},
"Additional sample data":{"RAW_FILE_NAME":"LCS_OE_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"LCS_KD_1",
"Factors":{"Treatment":"LCS silencing"},
"Additional sample data":{"RAW_FILE_NAME":"LCS_KD_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"LCS_KD_2",
"Factors":{"Treatment":"LCS silencing"},
"Additional sample data":{"RAW_FILE_NAME":"LCS_KD_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"LCS_KD_3",
"Factors":{"Treatment":"LCS silencing"},
"Additional sample data":{"RAW_FILE_NAME":"LCS_KD_3.raw"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Cells were washed in ice cold PBS and collected by scaling","SAMPLE_TYPE":"HeLa cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"Cells were treated to overexposes or down regulate GOLPH3 or LCS"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Samples were fortified with an Internal standard Mix and lipids were extracted twice with an extraction mix consisting of 85:15 Ethyl acetate 70% Isopropanol. All lipids that were used in the Internal standard mix and in the Calibration mixes were purchased from Avanti Polar Lipids Inc. After evaporating the cell extract to dryness, the samples were reconstituted in the mobile phase."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Samples were analyzed with a Quantum Ultra triple quadrupole mass spectrometer connected to an Accela HPLC and Accela autosampler using a solvent gradient. Ceramides identity was achieved through MRM analysis with soft fragmentation. Quantitative analysis is based on calibration curves generated for each ceramide. J. Bielawski et al. / Methods 39 (2006) 82–91","CHROMATOGRAPHY_TYPE":"Normal phase","INSTRUMENT_NAME":"Thermo Accela 1250","COLUMN_NAME":"Thermo Accucore C18 (100 x 2.1mm, 2.6um)"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Quantum Ultra","INSTRUMENT_TYPE":"Triple quadrupole","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"Samples were analyzed with a Quantum Ultra triple quadrupole mass spectrometer connected to an Accela HPLC and Accela autosampler using a solvent gradient. Ceramides identity was achieved through MRM analysis with soft fragmentation. Quantitative analysis is based on calibration curves generated for each ceramide. J. Bielawski et al. / Methods 39 (2006) 82–91"},

"MS_METABOLITE_DATA":{
"Units":"pmoles",

"Data":[{"Metabolite":"C14-Cer","CTRL_1":"10.524","CTRL_2":"12.143","CTRL_3":"12.777","GOLPH3_OE_1":"10.117","GOLPH3_OE_2":"10.099","GOLPH3_OE_3":"9.014","GOLPH3_KD_1":"18.467","GOLPH3_KD_2":"18.560","GOLPH3_KD_3":"20.692","LCS_OE_1":"10.103","LCS_OE_2":"9.377","LCS_OE_3":"7.842","LCS_KD_1":"16.024","LCS_KD_2":"14.685","LCS_KD_3":"14.985"},{"Metabolite":"C16-Cer","CTRL_1":"35.906","CTRL_2":"36.190","CTRL_3":"36.694","GOLPH3_OE_1":"28.833","GOLPH3_OE_2":"28.387","GOLPH3_OE_3":"25.984","GOLPH3_KD_1":"53.758","GOLPH3_KD_2":"53.089","GOLPH3_KD_3":"57.076","LCS_OE_1":"29.387","LCS_OE_2":"27.554","LCS_OE_3":"25.172","LCS_KD_1":"75.411","LCS_KD_2":"73.090","LCS_KD_3":"71.370"},{"Metabolite":"C18_1-Cer","CTRL_1":"5.293","CTRL_2":"6.152","CTRL_3":"6.728","GOLPH3_OE_1":"6.669","GOLPH3_OE_2":"6.475","GOLPH3_OE_3":"5.055","GOLPH3_KD_1":"6.503","GOLPH3_KD_2":"7.185","GOLPH3_KD_3":"8.165","LCS_OE_1":"5.860","LCS_OE_2":"5.768","LCS_OE_3":"4.900","LCS_KD_1":"4.583","LCS_KD_2":"4.410","LCS_KD_3":"4.411"},{"Metabolite":"C18-Cer","CTRL_1":"13.798","CTRL_2":"15.603","CTRL_3":"16.262","GOLPH3_OE_1":"15.493","GOLPH3_OE_2":"16.009","GOLPH3_OE_3":"12.584","GOLPH3_KD_1":"17.953","GOLPH3_KD_2":"20.969","GOLPH3_KD_3":"22.146","LCS_OE_1":"15.895","LCS_OE_2":"15.164","LCS_OE_3":"12.767","LCS_KD_1":"18.391","LCS_KD_2":"16.905","LCS_KD_3":"17.239"},{"Metabolite":"C20_1-Cer","CTRL_1":"2.632","CTRL_2":"2.999","CTRL_3":"3.182","GOLPH3_OE_1":"3.596","GOLPH3_OE_2":"3.200","GOLPH3_OE_3":"2.790","GOLPH3_KD_1":"4.749","GOLPH3_KD_2":"4.917","GOLPH3_KD_3":"5.433","LCS_OE_1":"3.462","LCS_OE_2":"2.935","LCS_OE_3":"2.590","LCS_KD_1":"2.557","LCS_KD_2":"2.223","LCS_KD_3":"2.339"},{"Metabolite":"C20-Cer","CTRL_1":"9.482","CTRL_2":"11.319","CTRL_3":"12.694","GOLPH3_OE_1":"13.820","GOLPH3_OE_2":"13.627","GOLPH3_OE_3":"9.976","GOLPH3_KD_1":"18.895","GOLPH3_KD_2":"21.744","GOLPH3_KD_3":"22.659","LCS_OE_1":"14.649","LCS_OE_2":"13.215","LCS_OE_3":"11.997","LCS_KD_1":"16.946","LCS_KD_2":"16.661","LCS_KD_3":"16.850"},{"Metabolite":"C22_1-Cer","CTRL_1":"43.438","CTRL_2":"46.563","CTRL_3":"45.329","GOLPH3_OE_1":"44.384","GOLPH3_OE_2":"42.803","GOLPH3_OE_3":"34.036","GOLPH3_KD_1":"77.131","GOLPH3_KD_2":"84.648","GOLPH3_KD_3":"91.685","LCS_OE_1":"42.912","LCS_OE_2":"41.757","LCS_OE_3":"36.849","LCS_KD_1":"30.979","LCS_KD_2":"30.198","LCS_KD_3":"30.714"},{"Metabolite":"C22-Cer","CTRL_1":"62.867","CTRL_2":"70.874","CTRL_3":"69.509","GOLPH3_OE_1":"80.418","GOLPH3_OE_2":"77.723","GOLPH3_OE_3":"58.186","GOLPH3_KD_1":"121.018","GOLPH3_KD_2":"147.954","GOLPH3_KD_3":"153.871","LCS_OE_1":"81.274","LCS_OE_2":"75.751","LCS_OE_3":"70.839","LCS_KD_1":"102.673","LCS_KD_2":"99.196","LCS_KD_3":"100.853"},{"Metabolite":"C24_1-Cer","CTRL_1":"222.961","CTRL_2":"254.854","CTRL_3":"265.953","GOLPH3_OE_1":"345.490","GOLPH3_OE_2":"347.029","GOLPH3_OE_3":"253.424","GOLPH3_KD_1":"530.947","GOLPH3_KD_2":"588.672","GOLPH3_KD_3":"637.582","LCS_OE_1":"364.076","LCS_OE_2":"341.059","LCS_OE_3":"311.230","LCS_KD_1":"442.159","LCS_KD_2":"444.161","LCS_KD_3":"427.374"},{"Metabolite":"C24-Cer","CTRL_1":"95.721","CTRL_2":"118.484","CTRL_3":"120.586","GOLPH3_OE_1":"135.123","GOLPH3_OE_2":"125.782","GOLPH3_OE_3":"94.763","GOLPH3_KD_1":"155.677","GOLPH3_KD_2":"185.417","GOLPH3_KD_3":"212.313","LCS_OE_1":"124.616","LCS_OE_2":"115.035","LCS_OE_3":"103.970","LCS_KD_1":"143.610","LCS_KD_2":"142.984","LCS_KD_3":"150.300"},{"Metabolite":"C26_1-Cer","CTRL_1":"3.185","CTRL_2":"3.834","CTRL_3":"3.817","GOLPH3_OE_1":"5.034","GOLPH3_OE_2":"4.817","GOLPH3_OE_3":"3.697","GOLPH3_KD_1":"6.466","GOLPH3_KD_2":"8.330","GOLPH3_KD_3":"9.349","LCS_OE_1":"4.793","LCS_OE_2":"4.697","LCS_OE_3":"3.950","LCS_KD_1":"5.126","LCS_KD_2":"5.167","LCS_KD_3":"5.076"},{"Metabolite":"C26-Cer","CTRL_1":"0.519","CTRL_2":"0.756","CTRL_3":"0.713","GOLPH3_OE_1":"1.212","GOLPH3_OE_2":"0.805","GOLPH3_OE_3":"0.590","GOLPH3_KD_1":"0.972","GOLPH3_KD_2":"1.339","GOLPH3_KD_3":"1.352","LCS_OE_1":"0.773","LCS_OE_2":"0.927","LCS_OE_3":"0.737","LCS_KD_1":"0.965","LCS_KD_2":"0.861","LCS_KD_3":"1.339"},{"Metabolite":"C14-SM","CTRL_1":"125.989","CTRL_2":"124.486","CTRL_3":"117.335","GOLPH3_OE_1":"119.393","GOLPH3_OE_2":"123.423","GOLPH3_OE_3":"87.996","GOLPH3_KD_1":"158.879","GOLPH3_KD_2":"172.220","GOLPH3_KD_3":"191.950","LCS_OE_1":"120.992","LCS_OE_2":"116.105","LCS_OE_3":"104.653","LCS_KD_1":"131.338","LCS_KD_2":"126.754","LCS_KD_3":"126.789"},{"Metabolite":"C16-SM","CTRL_1":"2189.630","CTRL_2":"2160.857","CTRL_3":"2171.885","GOLPH3_OE_1":"2273.539","GOLPH3_OE_2":"2314.236","GOLPH3_OE_3":"1671.577","GOLPH3_KD_1":"2541.305","GOLPH3_KD_2":"2998.993","GOLPH3_KD_3":"3180.184","LCS_OE_1":"2238.547","LCS_OE_2":"2270.450","LCS_OE_3":"1948.984","LCS_KD_1":"2953.994","LCS_KD_2":"2961.602","LCS_KD_3":"2761.761"},{"Metabolite":"C18_1-SM","CTRL_1":"60.593","CTRL_2":"58.002","CTRL_3":"56.037","GOLPH3_OE_1":"65.187","GOLPH3_OE_2":"62.212","GOLPH3_OE_3":"55.020","GOLPH3_KD_1":"60.336","GOLPH3_KD_2":"63.861","GOLPH3_KD_3":"65.415","LCS_OE_1":"62.046","LCS_OE_2":"60.30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}