#METABOLOMICS WORKBENCH Giovanni_DAngelo_20210129_093257 DATATRACK_ID:2436 STUDY_ID:ST001673 ANALYSIS_ID:AN002731 PROJECT_ID:PR001076
VERSION             	1
CREATED_ON             	February 2, 2021, 10:15 am
#PROJECT
PR:PROJECT_TITLE                 	Targeted Sphingolipid analysis of HeLa silenced for or overexpressing GOLPH3 or
PR:PROJECT_TITLE                 	LCS
PR:PROJECT_SUMMARY               	A group of sequentially-acting enzymes operating at the branchpoint among
PR:PROJECT_SUMMARY               	sphingolipid synthetic pathways binds the Golgi-localised oncoprotein GOLPH3.
PR:PROJECT_SUMMARY               	GOLPH3 sorts these enzymes into vesicles for intra-Golgi retro-transport, acting
PR:PROJECT_SUMMARY               	as a component of the cisternae inter-conversion mechanisms. Through these
PR:PROJECT_SUMMARY               	effects, GOLPH3 controls the sub-Golgi localisation, and the lysosomal
PR:PROJECT_SUMMARY               	degradation rate of specific enzymes. Here we evaluated the impact of
PR:PROJECT_SUMMARY               	overexpressing or silencing GOLPH3 or its client enzyme lactosylceramide
PR:PROJECT_SUMMARY               	synthase (LCS) on the sphingolipid composition of HeLa cells by targeted lipid
PR:PROJECT_SUMMARY               	analysis.
PR:INSTITUTE                     	École polytechnique fédérale de Lausanne (EPFL)
PR:DEPARTMENT                    	IBI
PR:LABORATORY                    	UPDANGELO
PR:LAST_NAME                     	D'Angelo
PR:FIRST_NAME                    	Giovanni
PR:ADDRESS                       	Station 15, Lausanne, Vaud, 1015, Switzerland
PR:EMAIL                         	giovanni.dangelo@epfl.ch
PR:PHONE                         	+41 216934276
#STUDY
ST:STUDY_TITLE                   	Targeted Sphingolipid analysis of HeLa silenced for or overexpressing GOLPH3 or
ST:STUDY_TITLE                   	LCS
ST:STUDY_SUMMARY                 	A group of sequentially-acting enzymes operating at the branchpoint among
ST:STUDY_SUMMARY                 	sphingolipid synthetic pathways binds the Golgi-localised oncoprotein GOLPH3.
ST:STUDY_SUMMARY                 	GOLPH3 sorts these enzymes into vesicles for intra-Golgi retro-transport, acting
ST:STUDY_SUMMARY                 	as a component of the cisternae inter-conversion mechanisms. Through these
ST:STUDY_SUMMARY                 	effects, GOLPH3 controls the sub-Golgi localisation, and the lysosomal
ST:STUDY_SUMMARY                 	degradation rate of specific enzymes. Here we evaluated the impact of
ST:STUDY_SUMMARY                 	overexpressing or silencing GOLPH3 or its client enzyme lactosylceramide
ST:STUDY_SUMMARY                 	synthase (LCS) on the sphingolipid composition of HeLa cells by targeted lipid
ST:STUDY_SUMMARY                 	analysis.
ST:INSTITUTE                     	École polytechnique fédérale de Lausanne (EPFL)
ST:DEPARTMENT                    	IBI
ST:LABORATORY                    	UPDANGELO
ST:LAST_NAME                     	D'Angelo
ST:FIRST_NAME                    	Giovanni
ST:ADDRESS                       	Station 15, Lausanne, Vaud, 1015, Switzerland
ST:EMAIL                         	giovanni.dangelo@epfl.ch
ST:PHONE                         	+41 216934276
#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]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	CTRL_1	Treatment:no treatment	RAW_FILE_NAME=CTRL_1.raw
SUBJECT_SAMPLE_FACTORS           	-	CTRL_2	Treatment:no treatment	RAW_FILE_NAME=CTRL_2.raw
SUBJECT_SAMPLE_FACTORS           	-	CTRL_3	Treatment:no treatment	RAW_FILE_NAME=CTRL_3.raw
SUBJECT_SAMPLE_FACTORS           	-	GOLPH3_OE_1	Treatment:GOLPH3 overexpression	RAW_FILE_NAME=GOLPH3_OE_1.raw
SUBJECT_SAMPLE_FACTORS           	-	GOLPH3_OE_2	Treatment:GOLPH3 overexpression	RAW_FILE_NAME=GOLPH3_OE_2.raw
SUBJECT_SAMPLE_FACTORS           	-	GOLPH3_OE_3	Treatment:GOLPH3 overexpression	RAW_FILE_NAME=GOLPH3_OE_3.raw
SUBJECT_SAMPLE_FACTORS           	-	GOLPH3_KD_1	Treatment:GOLPH3 silencing	RAW_FILE_NAME=GOLPH3_KD_1.raw
SUBJECT_SAMPLE_FACTORS           	-	GOLPH3_KD_2	Treatment:GOLPH3 silencing	RAW_FILE_NAME=GOLPH3_KD_2.raw
SUBJECT_SAMPLE_FACTORS           	-	GOLPH3_KD_3	Treatment:GOLPH3 silencing	RAW_FILE_NAME=GOLPH3_KD_3.raw
SUBJECT_SAMPLE_FACTORS           	-	LCS_OE_1	Treatment:LCS overexpression	RAW_FILE_NAME=LCS_OE_1.raw
SUBJECT_SAMPLE_FACTORS           	-	LCS_OE_2	Treatment:LCS overexpression	RAW_FILE_NAME=LCS_OE_2.raw
SUBJECT_SAMPLE_FACTORS           	-	LCS_OE_3	Treatment:LCS overexpression	RAW_FILE_NAME=LCS_OE_3.raw
SUBJECT_SAMPLE_FACTORS           	-	LCS_KD_1	Treatment:LCS silencing	RAW_FILE_NAME=LCS_KD_1.raw
SUBJECT_SAMPLE_FACTORS           	-	LCS_KD_2	Treatment:LCS silencing	RAW_FILE_NAME=LCS_KD_2.raw
SUBJECT_SAMPLE_FACTORS           	-	LCS_KD_3	Treatment:LCS silencing	RAW_FILE_NAME=LCS_KD_3.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	Cells were washed in ice cold PBS and collected by scaling
CO:SAMPLE_TYPE                   	HeLa cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Cells were treated to overexposes or down regulate GOLPH3 or LCS
#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:CHROMATOGRAPHY_TYPE           	Normal phase
CH:INSTRUMENT_NAME               	Thermo Accela 1250
CH:COLUMN_NAME                   	Thermo Accucore C18 (100 x 2.1mm, 2.6um)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Quantum Ultra
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
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_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	pmoles
MS_METABOLITE_DATA_START
Samples	CTRL_1	CTRL_2	CTRL_3	GOLPH3_OE_1	GOLPH3_OE_2	GOLPH3_OE_3	GOLPH3_KD_1	GOLPH3_KD_2	GOLPH3_KD_3	LCS_OE_1	LCS_OE_2	LCS_OE_3	LCS_KD_1	LCS_KD_2	LCS_KD_3
Factors	Treatment:no treatment	Treatment:no treatment	Treatment:no treatment	Treatment:GOLPH3 overexpression	Treatment:GOLPH3 overexpression	Treatment:GOLPH3 overexpression	Treatment:GOLPH3 silencing	Treatment:GOLPH3 silencing	Treatment:GOLPH3 silencing	Treatment:LCS overexpression	Treatment:LCS overexpression	Treatment:LCS overexpression	Treatment:LCS silencing	Treatment:LCS silencing	Treatment:LCS silencing
C14-Cer	10.524	12.143	12.777	10.117	10.099	9.014	18.467	18.560	20.692	10.103	9.377	7.842	16.024	14.685	14.985
C16-Cer	35.906	36.190	36.694	28.833	28.387	25.984	53.758	53.089	57.076	29.387	27.554	25.172	75.411	73.090	71.370
C18_1-Cer	5.293	6.152	6.728	6.669	6.475	5.055	6.503	7.185	8.165	5.860	5.768	4.900	4.583	4.410	4.411
C18-Cer	13.798	15.603	16.262	15.493	16.009	12.584	17.953	20.969	22.146	15.895	15.164	12.767	18.391	16.905	17.239
C20_1-Cer	2.632	2.999	3.182	3.596	3.200	2.790	4.749	4.917	5.433	3.462	2.935	2.590	2.557	2.223	2.339
C20-Cer	9.482	11.319	12.694	13.820	13.627	9.976	18.895	21.744	22.659	14.649	13.215	11.997	16.946	16.661	16.850
C22_1-Cer	43.438	46.563	45.329	44.384	42.803	34.036	77.131	84.648	91.685	42.912	41.757	36.849	30.979	30.198	30.714
C22-Cer	62.867	70.874	69.509	80.418	77.723	58.186	121.018	147.954	153.871	81.274	75.751	70.839	102.673	99.196	100.853
C24_1-Cer	222.961	254.854	265.953	345.490	347.029	253.424	530.947	588.672	637.582	364.076	341.059	311.230	442.159	444.161	427.374
C24-Cer	95.721	118.484	120.586	135.123	125.782	94.763	155.677	185.417	212.313	124.616	115.035	103.970	143.610	142.984	150.300
C26_1-Cer	3.185	3.834	3.817	5.034	4.817	3.697	6.466	8.330	9.349	4.793	4.697	3.950	5.126	5.167	5.076
C26-Cer	0.519	0.756	0.713	1.212	0.805	0.590	0.972	1.339	1.352	0.773	0.927	0.737	0.965	0.861	1.339
C14-SM	125.989	124.486	117.335	119.393	123.423	87.996	158.879	172.220	191.950	120.992	116.105	104.653	131.338	126.754	126.789
C16-SM	2189.630	2160.857	2171.885	2273.539	2314.236	1671.577	2541.305	2998.993	3180.184	2238.547	2270.450	1948.984	2953.994	2961.602	2761.761
C18_1-SM	60.593	58.002	56.037	65.187	62.212	55.020	60.336	63.861	65.415	62.046	60.300	57.506	50.576	48.620	52.123
C18-SM	196.435	188.565	187.946	221.096	219.259	190.059	250.601	250.205	272.089	213.609	210.508	208.144	240.290	242.502	235.196
C20_1-SM	26.175	24.180	25.393	30.744	28.933	24.702	39.004	41.390	42.977	28.516	27.575	27.788	20.993	19.657	21.102
C20-SM	57.936	55.959	52.021	70.342	67.992	52.491	85.369	84.106	96.504	58.543	60.646	59.618	58.183	63.313	57.461
C22_1-SM	364.891	343.716	333.969	379.379	385.383	312.193	521.290	538.664	590.070	346.405	349.420	335.217	210.657	196.203	204.957
C22-SM	272.917	269.672	268.882	291.876	275.171	240.530	340.334	363.971	402.325	249.701	267.316	241.777	211.192	202.985	209.654
C24_1-SM	2552.009	2452.597	2442.822	2717.197	2489.529	2243.303	3732.953	3584.005	3880.436	2434.598	2451.226	2286.429	2132.060	2093.137	2115.014
C24-SM	235.420	227.654	237.527	204.753	194.967	189.821	272.453	272.515	283.615	170.912	184.293	157.975	166.493	171.659	164.654
C26_1-SM	17.332	16.806	17.882	14.327	13.226	13.145	28.571	23.060	28.466	11.825	12.989	13.071	11.987	12.539	12.858
C26-SM	2.528	2.471	2.623	2.500	2.201	2.183	2.741	2.799	2.918	2.308	2.220	2.319	2.294	2.381	2.367
C14Lact-Cer	0.551	BQL	BQL	BQL	BQL	BQL	BQL	BQL	BQL	BQL	BQL	BQL	0.489	0.487	BQL
C14HexosylCer	3.127	3.622	3.522	4.246	4.226	3.720	5.931	6.010	6.670	3.514	3.196	3.182	10.524	9.775	8.758
C16Lact-Cer	38.100	43.091	43.928	70.063	67.540	49.720	52.277	55.532	65.711	88.354	86.098	77.279	29.670	27.331	28.698
C18_1Lact-Cer	0.736	0.744	0.801	0.922	0.883	0.831	1.151	0.940	1.075	1.157	1.159	0.943	0.703	0.658	0.724
C16HexosylCer	75.348	88.958	84.562	110.325	111.280	91.754	132.145	144.953	150.342	81.072	78.255	71.280	443.144	426.484	367.825
C18_1HexosylCer	1.435	1.513	1.504	1.554	1.583	1.486	1.589	1.673	1.630	1.500	1.496	1.502	1.447	1.687	1.557
C18Lact-Cer	6.074	7.236	7.331	9.415	9.186	8.200	10.617	9.966	10.973	14.157	13.964	12.671	4.968	4.075	4.384
C20_1Lact-Cer	1.063	1.126	1.019	1.145	1.185	1.111	1.534	1.549	1.503	1.813	1.620	1.617	0.845	0.740	0.767
C18HexosylCer	7.531	7.650	7.701	10.252	9.712	9.729	10.838	11.506	11.028	8.136	7.891	7.658	36.649	35.823	31.098
C20_1HexosylCer	1.994	2.090	1.970	2.837	2.596	2.473	4.095	3.863	3.864	2.284	2.293	2.161	3.529	3.441	3.189
C20Lact-Cer	2.062	2.208	2.099	2.705	2.472	2.265	2.713	2.657	2.396	4.024	4.110	3.503	1.482	1.403	1.518
C22_1Lact-Cer	6.127	5.785	6.172	7.895	6.526	6.711	10.419	9.822	9.851	13.491	11.900	12.188	3.528	3.553	3.618
C20HexosylCer	5.521	5.232	5.383	7.090	7.397	7.192	7.763	8.361	8.087	5.771	5.544	5.484	18.292	17.258	16.105
C22_1HexosylCer	13.494	15.214	15.497	23.720	23.931	21.868	41.887	39.407	41.357	17.882	17.987	16.937	31.812	29.859	26.668
C22Lact-Cer	30.934	34.347	36.028	36.460	34.082	33.514	44.877	44.596	51.805	59.259	57.279	54.881	13.072	13.359	13.436
C24_1Lact-Cer	105.750	107.947	112.621	148.376	135.023	133.263	177.886	174.459	183.727	243.061	219.416	213.961	56.490	53.871	59.014
C22HexosylCer	162.235	184.529	191.004	223.450	230.233	201.509	293.005	303.590	314.592	178.398	176.450	161.947	485.336	440.644	404.053
C24_1HexosylCer	454.074	501.389	512.099	705.367	695.840	607.689	878.620	931.960	961.134	584.663	566.360	525.635	1256.146	1172.306	1050.384
C24Lact-Cer	41.135	43.646	44.472	51.809	48.747	44.518	47.979	47.591	48.766	71.842	69.486	66.921	20.702	19.995	21.360
C26_1Lact-Cer	4.942	5.020	4.672	5.015	5.039	4.568	5.849	6.044	6.058	6.822	6.496	6.497	3.281	3.421	3.332
C24HexosylCer	253.848	324.527	330.981	369.630	363.637	287.918	445.579	464.222	499.899	281.578	277.578	242.540	724.827	648.820	571.066
C26_1HexosylCer	20.396	23.607	22.593	23.189	23.723	19.608	32.433	33.722	35.256	18.992	18.996	17.562	35.502	33.050	27.579
C26Lact-Cer	2.156	2.282	2.301	2.319	2.307	2.257	2.379	2.624	2.579	2.493	2.489	2.375	2.092	2.094	2.063
C26HexosylCer	3.479	4.023	4.028	4.133	4.179	3.750	4.076	4.654	4.815	3.543	3.463	3.519	4.723	4.643	4.713
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	RT
C14-Cer	15.053
C16-Cer	16.57993424
C18_1-Cer	17.0608333
C18-Cer	17.93720568
C20_1-Cer	18.62231951
C20-Cer	19.3108956
C22_1-Cer	19.94082869
C22-Cer	20.77011827
C24_1-Cer	20.97331072
C24-Cer	22.3756384
C26_1-Cer	22.87423635
C26-Cer	24.53351824
C14-SM	17.662
C16-SM	18.481
C18_1-SM	19.081
C18-SM	19.711
C20_1-SM	19.964
C20-SM	20.845
C22_1-SM	20.658
C22-SM	21.602
C24_1-SM	21.729
C24-SM	22.988
C26_1-SM	23.083
C26-SM	24.624
C14Lact-Cer	15.552
C14HexosylCer	15.979
C16Lact-Cer	16.742
C18_1Lact-Cer	16.744
C16HexosylCer	17.160
C18_1HexosylCer	17.550
C20_1Lact-Cer	17.808
C18Lact-Cer	17.804
C18HexosylCer	18.167
C20_1HexosylCer	18.170
C20Lact-Cer	18.814
C22_1Lact-Cer	19.037
C20HexosylCer	19.233
C22_1HexosylCer	19.455
C22Lact-Cer	19.875
C24_1Lact-Cer	20.046
C22HexosylCer	20.404
C24_1HexosylCer	20.576
C26_1Lact-Cer	21.109
C24Lact-Cer	21.866
C26_1HexosylCer	21.748
C24HexosylCer	22.562
C26Lact-Cer	22.561
C26HexosylCer	23.258
METABOLITES_END
#END