{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001877","ANALYSIS_ID":"AN003080","VERSION":"1","CREATED_ON":"03-03-2022"},

"PROJECT":{"PROJECT_TITLE":"Targeted Sphingolipid analysis of HeLa knockout for expression of GRASP55","PROJECT_SUMMARY":"Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 acts by binding to these enzymes and preventing their entry to COPI derived retrograde transport vesicles thus concentrating them in the trans-Golgi. In genome edited cells lacking GRASP55 the enzymes relocate to cis-Golgi. Here we evaluated the impact of deleting GRASP55 on sphingolipid composition of HeLa cells by targeted lipid analysis.","INSTITUTE":"IBBC, CNR","LAST_NAME":"parashuraman","FIRST_NAME":"seetharaman","ADDRESS":"Via Pietro Castellino 111, Napoli, NA, 80131, Italy","EMAIL":"raman@ibbc.cnr.it","PHONE":"0816132283","DOI":"http://dx.doi.org/10.21228/M8SM4R"},

"STUDY":{"STUDY_TITLE":"Targeted Sphingolipid analysis of HeLa knockout for expression of GRASP55","STUDY_SUMMARY":"Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 acts by binding to these enzymes and preventing their entry to COPI derived retrograde transport vesicles thus concentrating them in the trans-Golgi. In genome edited cells lacking GRASP55 the enzymes relocate to cis-Golgi. Here we evaluated the impact of deleting GRASP55 on sphingolipid composition of HeLa cells by targeted lipid analysis.","INSTITUTE":"IBBC, CNR","LAST_NAME":"parashuraman","FIRST_NAME":"seetharaman","ADDRESS":"Via Pietro Castellino 111, Napoli, NA, 80131, Italy","EMAIL":"raman@ibbc.cnr.it","PHONE":"0816132283","SUBMIT_DATE":"2021-07-18"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"KO_1",
"Factors":{"Treatment":"GRASP55 KO"},
"Additional sample data":{"RAW_FILE_NAME":"RA-7.raw"}
},
{
"Subject ID":"-",
"Sample ID":"KO_2",
"Factors":{"Treatment":"GRASP55 KO"},
"Additional sample data":{"RAW_FILE_NAME":"RA-8.raw"}
},
{
"Subject ID":"-",
"Sample ID":"KO_3",
"Factors":{"Treatment":"GRASP55 KO"},
"Additional sample data":{"RAW_FILE_NAME":"RA-9.raw"}
},
{
"Subject ID":"-",
"Sample ID":"C_1",
"Factors":{"Treatment":"no treatment"},
"Additional sample data":{"RAW_FILE_NAME":"RA-1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"C_2",
"Factors":{"Treatment":"no treatment"},
"Additional sample data":{"RAW_FILE_NAME":"RA-2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"C_3",
"Factors":{"Treatment":"no treatment"},
"Additional sample data":{"RAW_FILE_NAME":"RA-3.raw"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Cells were washed in ice cold PBS and collected by scraping","SAMPLE_TYPE":"HeLa cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"Cells were treated with CRISPR/Cas9 to knockout GRASP55 expression"},

"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","INSTRUMENT_NAME":"Thermo Accela 1250","COLUMN_NAME":"Thermo Accucore C18 (100 x 2.1mm, 2.6um)","CHROMATOGRAPHY_TYPE":"Reversed phase"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Quantum Ultra","INSTRUMENT_TYPE":"Triple quadrupole","MS_TYPE":"ESI","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","ION_MODE":"POSITIVE"},

"MS_METABOLITE_DATA":{
"Units":"pmole/nmole Pi",

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}

}