#METABOLOMICS WORKBENCH raman_20210718_232851 DATATRACK_ID:2753 STUDY_ID:ST001877 ANALYSIS_ID:AN003080 VERSION 1 CREATED_ON 03-03-2022 #PROJECT PR:PROJECT_TITLE Targeted Sphingolipid analysis of HeLa knockout for expression of GRASP55 PR:PROJECT_SUMMARY Golgi apparatus, the main glycosylation station of the cell, consists of a stack PR:PROJECT_SUMMARY of discontinuous cisternae. Glycosylation enzymes are usually concentrated in PR:PROJECT_SUMMARY one or two specific cisternae along the cis-trans axis of the organelle. How PR:PROJECT_SUMMARY such compartmentalized localization of enzymes is achieved and how it PR:PROJECT_SUMMARY contributes to glycosylation are not clear. Here we show that the Golgi matrix PR:PROJECT_SUMMARY protein GRASP55 directs the compartmentalized localization of key enzymes PR:PROJECT_SUMMARY involved in glycosphingolipid (GSL) biosynthesis. GRASP55 acts by binding to PR:PROJECT_SUMMARY these enzymes and preventing their entry to COPI derived retrograde transport PR:PROJECT_SUMMARY vesicles thus concentrating them in the trans-Golgi. In genome edited cells PR:PROJECT_SUMMARY lacking GRASP55 the enzymes relocate to cis-Golgi. Here we evaluated the impact PR:PROJECT_SUMMARY of deleting GRASP55 on sphingolipid composition of HeLa cells by targeted lipid PR:PROJECT_SUMMARY analysis. PR:INSTITUTE IBBC, CNR PR:LAST_NAME parashuraman PR:FIRST_NAME seetharaman PR:ADDRESS Via Pietro Castellino 111, Napoli, NA, 80131, Italy PR:EMAIL raman@ibbc.cnr.it PR:PHONE 0816132283 PR:DOI http://dx.doi.org/10.21228/M8SM4R #STUDY ST:STUDY_TITLE Targeted Sphingolipid analysis of HeLa knockout for expression of GRASP55 ST:STUDY_SUMMARY Golgi apparatus, the main glycosylation station of the cell, consists of a stack ST:STUDY_SUMMARY of discontinuous cisternae. Glycosylation enzymes are usually concentrated in ST:STUDY_SUMMARY one or two specific cisternae along the cis-trans axis of the organelle. How ST:STUDY_SUMMARY such compartmentalized localization of enzymes is achieved and how it ST:STUDY_SUMMARY contributes to glycosylation are not clear. Here we show that the Golgi matrix ST:STUDY_SUMMARY protein GRASP55 directs the compartmentalized localization of key enzymes ST:STUDY_SUMMARY involved in glycosphingolipid (GSL) biosynthesis. GRASP55 acts by binding to ST:STUDY_SUMMARY these enzymes and preventing their entry to COPI derived retrograde transport ST:STUDY_SUMMARY vesicles thus concentrating them in the trans-Golgi. In genome edited cells ST:STUDY_SUMMARY lacking GRASP55 the enzymes relocate to cis-Golgi. Here we evaluated the impact ST:STUDY_SUMMARY of deleting GRASP55 on sphingolipid composition of HeLa cells by targeted lipid ST:STUDY_SUMMARY analysis. ST:INSTITUTE IBBC, CNR ST:LAST_NAME parashuraman ST:FIRST_NAME seetharaman ST:ADDRESS Via Pietro Castellino 111, Napoli, NA, 80131, Italy ST:EMAIL raman@ibbc.cnr.it ST:PHONE 0816132283 ST:SUBMIT_DATE 2021-07-18 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - KO_1 Treatment:GRASP55 KO RAW_FILE_NAME=RA-7.raw SUBJECT_SAMPLE_FACTORS - KO_2 Treatment:GRASP55 KO RAW_FILE_NAME=RA-8.raw SUBJECT_SAMPLE_FACTORS - KO_3 Treatment:GRASP55 KO RAW_FILE_NAME=RA-9.raw SUBJECT_SAMPLE_FACTORS - C_1 Treatment:no treatment RAW_FILE_NAME=RA-1.raw SUBJECT_SAMPLE_FACTORS - C_2 Treatment:no treatment RAW_FILE_NAME=RA-2.raw SUBJECT_SAMPLE_FACTORS - C_3 Treatment:no treatment RAW_FILE_NAME=RA-3.raw #COLLECTION CO:COLLECTION_SUMMARY Cells were washed in ice cold PBS and collected by scraping CO:SAMPLE_TYPE HeLa cells #TREATMENT TR:TREATMENT_SUMMARY Cells were treated with CRISPR/Cas9 to knockout GRASP55 expression #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were fortified with an Internal standard Mix and lipids were extracted SP:SAMPLEPREP_SUMMARY twice with an extraction mix consisting of 85:15 Ethyl acetate 70% Isopropanol. SP:SAMPLEPREP_SUMMARY All lipids that were used in the Internal standard mix and in the Calibration SP:SAMPLEPREP_SUMMARY mixes were purchased from Avanti Polar Lipids Inc. After evaporating the cell SP:SAMPLEPREP_SUMMARY extract to dryness, the samples were reconstituted in the mobile phase. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples were analyzed with a Quantum Ultra triple quadrupole mass spectrometer CH:CHROMATOGRAPHY_SUMMARY connected to an Accela HPLC and Accela autosampler using a solvent gradient. CH:CHROMATOGRAPHY_SUMMARY Ceramides identity was achieved through MRM analysis with soft fragmentation. CH:CHROMATOGRAPHY_SUMMARY Quantitative analysis is based on calibration curves generated for each CH:CHROMATOGRAPHY_SUMMARY ceramide. J. Bielawski et al. / Methods 39 (2006) 82–91 CH:INSTRUMENT_NAME Thermo Accela 1250 CH:COLUMN_NAME Thermo Accucore C18 (100 x 2.1mm, 2.6um) CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Quantum Ultra MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:MS_COMMENTS Samples were analyzed with a Quantum Ultra triple quadrupole mass spectrometer MS:MS_COMMENTS connected to an Accela HPLC and Accela autosampler using a solvent gradient. MS:MS_COMMENTS Ceramides identity was achieved through MRM analysis with soft fragmentation. MS:MS_COMMENTS Quantitative analysis is based on calibration curves generated for each MS:MS_COMMENTS ceramide. J. Bielawski et al. / Methods 39 (2006) 82–91 MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS pmole/nmole Pi MS_METABOLITE_DATA_START Samples KO_1 KO_2 KO_3 C_1 C_2 C_3 Factors Treatment:GRASP55 KO Treatment:GRASP55 KO Treatment:GRASP55 KO Treatment:no treatment Treatment:no treatment Treatment:no treatment C14-Cer 0.1068 0.1539 0.1475 0.1075 0.1000 0.1338 C14HexosylCer 0.0369 0.0563 0.0546 0.0457 0.0430 0.0494 C14Lact-Cer 0.0494 0.0608 0.0685 0.0488 0.0486 0.0590 C14-SM 4.3003 5.6899 5.8460 4.5049 4.8950 5.4791 C16-Cer 0.2844 0.4004 0.3703 0.2459 0.2170 0.2783 C16HexosylCer 0.8669 1.3134 1.2666 1.1375 1.0284 1.3019 C16Lact-Cer 0.6286 0.8845 0.8712 0.7623 0.7268 0.9100 C16-SM 103.7501 141.7738 148.7819 123.4809 127.5306 139.2992 C18_1-Cer 0.0313 0.0407 0.0443 0.0359 0.0371 0.0376 C18_1Lact-Cer 0.0589 0.0813 0.0810 0.0601 0.0592 0.0742 C18_1-SM 0.5056 0.7008 0.6577 0.5147 0.5069 0.6023 C18-Cer 0.0719 0.0982 0.1085 0.0585 0.0602 0.0618 C18HexosylCer 0.1345 0.1823 0.1748 0.0983 0.0873 0.1162 C18Lact-Cer 0.1053 0.1556 0.1446 0.1185 0.0991 0.1315 C18-SM 2.8332 3.8949 3.6631 2.7048 2.6868 3.1902 C20_1-Cer 0.0153 0.0179 0.0224 0.0133 0.0154 0.0139 C20_1HexosylCer 0.0189 0.0239 0.0213 0.0118 0.0119 0.0151 C20_1Lact-Cer 0.0208 0.0256 0.0219 0.0113 0.0086 0.0167 C20_1-SM 0.2260 0.3070 0.2875 0.2201 0.2163 0.2641 C20-Cer 0.0938 0.1233 0.1458 0.1023 0.0964 0.0992 C20HexosylCer 0.0830 0.1165 0.0963 0.0464 0.0384 0.0505 C20Lact-Cer 0.0366 0.0548 0.0419 0.0329 0.0245 0.0327 C20-SM 0.9182 1.2731 1.2139 0.9171 0.9163 1.0793 C22_1-Cer 0.0533 0.0659 0.0632 0.0288 0.0308 0.0381 C22_1HexosylCer 0.1421 0.2145 0.1992 0.0807 0.0769 0.1041 C22_1Lact-Cer 0.0791 0.0990 0.0784 0.0316 0.0282 0.0390 C22_1-SM 2.4352 3.4182 3.1915 2.6916 2.7216 3.2153 C22-Cer 0.2179 0.2956 0.2782 0.2207 0.2336 0.2851 C22HexosylCer 1.5929 2.3615 2.5108 1.6046 1.4165 2.0185 C22Lact-Cer 0.3689 0.4872 0.4654 0.2901 0.2640 0.3706 C22-SM 9.7186 13.9209 12.8455 11.5385 11.3735 13.8262 C24_1-Cer 0.7472 1.0443 0.9924 0.7183 0.7884 0.9574 C24_1HexosylCer 3.7755 5.5058 5.8885 3.1002 2.7740 4.0357 C24_1Lact-Cer 1.2919 1.8969 1.9318 0.8377 0.7787 1.0928 C24_1-SM 10.9742 15.2878 14.6088 14.3059 14.7179 17.3668 C24-Cer 0.3105 0.4261 0.3942 0.5143 0.5586 0.6731 C24HexosylCer 2.0677 3.0403 3.5600 3.0291 2.9259 4.3627 C24Lact-Cer 0.6441 0.8431 1.0178 0.6353 0.6550 0.8517 C24-SM 4.4757 6.2707 5.8897 6.2944 6.1819 7.4532 C26_1-Cer 0.0177 0.0249 0.0223 0.0186 0.0210 0.0251 C26_1HexosylCer 0.1703 0.2389 0.2687 0.1761 0.1620 0.2420 C26_1Lact-Cer 0.0463 0.0622 0.0676 0.0218 0.0229 0.0320 C26_1-SM 0.1084 0.1444 0.1354 0.0971 0.0970 0.1185 C26-Cer 0.0035 0.0047 0.0045 0.0056 0.0062 0.0071 C26HexosylCer 0.0284 0.0385 0.0391 0.0327 0.0339 0.0478 C26Lact-Cer 0.0120 0.0184 0.0193 0.0116 0.0109 0.0160 C26-SM 0.0343 0.0531 0.0431 0.0339 0.0313 0.0428 Hexosyl-Sph 0.0018 0.0023 0.0025 0.0030 0.0032 0.0039 Lact-Sph 0.0017 0.0025 0.0032 0.0020 0.0022 0.0019 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant C14-Cer C14HexosylCer C14Lact-Cer C14-SM C16-Cer C16HexosylCer C16Lact-Cer C16-SM C18_1-Cer C18_1Lact-Cer C18_1-SM C18-Cer C18HexosylCer C18Lact-Cer C18-SM C20_1-Cer C20_1HexosylCer C20_1Lact-Cer C20_1-SM C20-Cer C20HexosylCer C20Lact-Cer C20-SM C22_1-Cer C22_1HexosylCer C22_1Lact-Cer C22_1-SM C22-Cer C22HexosylCer C22Lact-Cer C22-SM C24_1-Cer C24_1HexosylCer C24_1Lact-Cer C24_1-SM C24-Cer C24HexosylCer C24Lact-Cer C24-SM C26_1-Cer C26_1HexosylCer C26_1Lact-Cer C26_1-SM C26-Cer C26HexosylCer C26Lact-Cer C26-SM Hexosyl-Sph Lact-Sph METABOLITES_END #END