#METABOLOMICS WORKBENCH ppjutur_20220622_232801 DATATRACK_ID:3315 STUDY_ID:ST002215 ANALYSIS_ID:AN003624 PROJECT_ID:PR001415
VERSION             	1
CREATED_ON             	July 11, 2022, 9:35 pm
#PROJECT
PR:PROJECT_TITLE                 	Multi-omics analysis of Aurantiochytrium limacinum
PR:PROJECT_TYPE                  	Time course GCMS based untargeted metabolomics in the presence of glucose and
PR:PROJECT_TYPE                  	glycerol
PR:PROJECT_SUMMARY               	To evaluate temporal changes in the presence of glycerol, time-course metabolite
PR:PROJECT_SUMMARY               	profiling was done in the presence of glycerol and glucose at three different
PR:PROJECT_SUMMARY               	time-points i.e., 0, 48 and 96 h using GCMS.
PR:INSTITUTE                     	International Centre for Genetic Engineering and Biotechnology
PR:DEPARTMENT                    	Omics of Algae
PR:LAST_NAME                     	Jutur
PR:FIRST_NAME                    	Pannaga Pavan
PR:ADDRESS                       	Omics of Algae Group, ICGEB campus, Aruna Asaf Ali Marg, New Delhi, Delhi,
PR:ADDRESS                       	110070, India
PR:EMAIL                         	jppavan@icgeb.res.in
PR:PHONE                         	26781358
#STUDY
ST:STUDY_TITLE                   	Time course GCMS based untargeted metabolomics in the presence of glucose and
ST:STUDY_TITLE                   	glycerol
ST:STUDY_SUMMARY                 	Our preliminary analysis identifies that glycerol enhanced DHA accumulation in
ST:STUDY_SUMMARY                 	native isolate Aurantiochytrium limacinum. To evaluate temporal changes in the
ST:STUDY_SUMMARY                 	presence of glycerol, time-course metabolite profiling was done in the presence
ST:STUDY_SUMMARY                 	of glycerol and glucose at three different time-points i.e., 0, 48 and 96 h
ST:STUDY_SUMMARY                 	using GC-MS. We identified nearly 40 metabolites, among which metabolites
ST:STUDY_SUMMARY                 	belonging to pentose phosphate pathway, TCA cycle and amino acid metabolism were
ST:STUDY_SUMMARY                 	significantly differentially regulated which suggests its role in glycerol
ST:STUDY_SUMMARY                 	induced DHA accumulation.
ST:INSTITUTE                     	International Centre for Genetic Engineering and Biotechnology
ST:LAST_NAME                     	Jutur
ST:FIRST_NAME                    	Pannaga Pavan
ST:ADDRESS                       	Omics of Algae Lab, 2nd Floor, New Building, ICGEB, Aruna Asaf Ali Marg, New
ST:ADDRESS                       	Delhi
ST:EMAIL                         	jppavan@gmail.com
ST:PHONE                         	01126741358
#SUBJECT
SU:SUBJECT_TYPE                  	Other organism
SU:SUBJECT_SPECIES               	Aurantiochytrium limacinum
#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           	-	D0_1	Treatment:Glucose	RAW_FILE_NAME=D0_1; Time point=0 h
SUBJECT_SAMPLE_FACTORS           	-	D0_2	Treatment:Glucose	RAW_FILE_NAME=D0_2; Time point=0 h
SUBJECT_SAMPLE_FACTORS           	-	D0_3	Treatment:Glucose	RAW_FILE_NAME=D0_3; Time point=0 h
SUBJECT_SAMPLE_FACTORS           	-	D2_Glucose_1	Treatment:Glucose	RAW_FILE_NAME=D2_GLU_1; Time point=48 h
SUBJECT_SAMPLE_FACTORS           	-	D2_Glucose_2	Treatment:Glucose	RAW_FILE_NAME=D2_GLU_2; Time point=48 h
SUBJECT_SAMPLE_FACTORS           	-	D2_Glucose_3	Treatment:Glucose	RAW_FILE_NAME=D2_GLU_3; Time point=48 h
SUBJECT_SAMPLE_FACTORS           	-	D2_Glycerol_1	Treatment:Glycerol	RAW_FILE_NAME=D2_GLY_1; Time point=48 h
SUBJECT_SAMPLE_FACTORS           	-	D2_Glycerol_2	Treatment:Glycerol	RAW_FILE_NAME=D2_GLY_2; Time point=48 h
SUBJECT_SAMPLE_FACTORS           	-	D2_Glycerol_3	Treatment:Glycerol	RAW_FILE_NAME=D2_GLY_3; Time point=48 h
SUBJECT_SAMPLE_FACTORS           	-	D4_Glucose_1	Treatment:Glucose	RAW_FILE_NAME=D4_GLU_1; Time point=96 h
SUBJECT_SAMPLE_FACTORS           	-	D4_Glucose_2	Treatment:Glucose	RAW_FILE_NAME=D4_GLU_2; Time point=96 h
SUBJECT_SAMPLE_FACTORS           	-	D4_Glucose_3	Treatment:Glucose	RAW_FILE_NAME=D4_GLU_3; Time point=96 h
SUBJECT_SAMPLE_FACTORS           	-	D4_Glycerol_1	Treatment:Glycerol	RAW_FILE_NAME=D4_GLY_1; Time point=96 h
SUBJECT_SAMPLE_FACTORS           	-	D4_Glycerol_2	Treatment:Glycerol	RAW_FILE_NAME=D4_GLY_2; Time point=96 h
SUBJECT_SAMPLE_FACTORS           	-	D4_Glycerol_3	Treatment:Glycerol	RAW_FILE_NAME=D4_GLY_3; Time point=96 h
#COLLECTION
CO:COLLECTION_SUMMARY            	Marine thraustochytrid Aurantiochytrium limacinum was a native isolate obtained
CO:COLLECTION_SUMMARY            	as a generous gift from ICT-Mumbai, India. It was cultivated in YPD medium with
CO:COLLECTION_SUMMARY            	the optimized concentration of 3% (w/v) glucose and glycerol at 20 C for a
CO:COLLECTION_SUMMARY            	duration of 96 h.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	It was cultivated in YPD medium with the optimized concentration of 3% (w/v)
TR:TREATMENT_SUMMARY             	glucose and glycerol at 20 C for a duration of 96 h.
TR:TREATMENT                     	Glucose and Glycerol
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Quenched cells were resuspended in 1 mL of ice-cold
SP:SAMPLEPREP_SUMMARY            	methanol/ethanol/chloroform(2:6:2), followed by sonication of resuspended cells
SP:SAMPLEPREP_SUMMARY            	in sonication bath for 15 min. Later, these samples were centrifuged at
SP:SAMPLEPREP_SUMMARY            	10,000×g for 15 min at 4 °C to get rid of cell debris. The supernatant was
SP:SAMPLEPREP_SUMMARY            	filtered using a 0.2-µm filter. One hundred microlitres of supernatant was
SP:SAMPLEPREP_SUMMARY            	taken and dried under nitrogen stream. The dried leftover was dissolved in 10
SP:SAMPLEPREP_SUMMARY            	µL of freshly prepared methoxyamine hydrochloride solution (40 mg mL−1 in
SP:SAMPLEPREP_SUMMARY            	pyridine) and incubated at 30 °C for 90 min with shaking. To the above
SP:SAMPLEPREP_SUMMARY            	solution, 90 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide was added and
SP:SAMPLEPREP_SUMMARY            	incubated at 37 °C for 30 min. The samples were centrifuged at 14,000×g for 3
SP:SAMPLEPREP_SUMMARY            	min, and the supernatant was taken for the GC-MS/MS analysis.
SP:PROCESSING_STORAGE_CONDITIONS 	Described in summary
SP:SAMPLE_DERIVATIZATION         	MSTFA
SP:SAMPLE_SPIKING                	Nonadecanoic acid
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	GC
CH:INSTRUMENT_NAME               	Agilent 7890A
CH:COLUMN_NAME                   	Agilent DB5-MS (30m x 0.25mm, 0.25um)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 7890A
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Mass Hunter
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Area
MS_METABOLITE_DATA_START
Samples	D0_1	D0_2	D0_3	D2_Glucose_1	D2_Glucose_2	D2_Glucose_3	D2_Glycerol_1	D2_Glycerol_2	D2_Glycerol_3	D4_Glucose_1	D4_Glucose_2	D4_Glucose_3	D4_Glycerol_1	D4_Glycerol_2	D4_Glycerol_3
Factors	Treatment:Glucose	Treatment:Glucose	Treatment:Glucose	Treatment:Glucose	Treatment:Glucose	Treatment:Glucose	Treatment:Glycerol	Treatment:Glycerol	Treatment:Glycerol	Treatment:Glucose	Treatment:Glucose	Treatment:Glucose	Treatment:Glycerol	Treatment:Glycerol	Treatment:Glycerol
L-Alanine	125584230.9	91734745.32	57885259.73	1382455.341	2626638.464		1244952.062	3228913.217	2236932.639		4188827.146	4188827.146	4094929.239		1589219.155
L-Glycine	88090719.42	59750586.54	31410453.66	605770.5	2038907.639		702385.6813	498136.4816	600261.0814		1742410.382	188200.8927	1044998.1		598763.5661
L-Valine	75521276.58	50716358.89	25911441.19	8075194.273	15305042.26		3240149.909	27329693.73	15284921.82		989737.614	989737.614	86405.66364		224068.6007
L-Leucine	78449500.5	52778231.47	27106962.45	579610.2955	2490625.663		395243.8687	3472645.97	1933944.919		1049757.27	1049757.27			
L-Proline	401234072.8	279133220.9	157032369	15597033.27	34996398.67		7712811.738	29865089.86	18788950.8		1805109.995	1805109.995	27034357.43		11650607.82
L-Isoleucine	27207072.54	18304945.44	9402818.335												
L-Serine	15133808.11	9973457.471	4813106.833	214328.4091	136933.3183		131706.819								
L-Threonine	71316512.99	47669495.77	24022478.56	793121.6136	999846.9987										
L-Methionine			1204881.242	41925.36364			73848.11165	533386.3947	303617.2532						
L-Phenylalanine			6524012.076	122660.7273				501949.1534					125646.9449		
L-Aspartic acid				49213.11364	119115.656		102639.543	394479.2061	248559.3745				444292.4996		
L-Tyrosine				816629.4091			559006.7837								
L-Tryptophan							18619.59805								
GABA	4276471.794			35748			59566.32511	253693.6292	156629.9772						
Propanoic acid	1378130.731			36096.29545		985772.6609					41519.65162	185605.2758			
Acetic acid	2347838.8	2522998.347	2698157.893		133275.8896			386441.7768			94569.56495	94569.56495			
Succinic acid	213616367.6	147621691.3	81627015.08	3330210.114	7158739.936		4071620.658	270516.2307	2171068.444		20215670.19		681323.8504		20364444.45
Malic acid	7023505.771	4441126.162	1858746.552	87664.77273											
Glutamic acid	1147070.969				70758.08197										
Phosphoric acid	2020629.832	1241195.636	461761.4395	111098.0682	5197164.124		209145.2268	478980.0588	344062.6428		3690062.257	3690062.257			
Glycerol	6215130.805	4329392.734	2443654.663			703080.5893		155244.7933							
Propanedioic acid	3192922.592	2087485.279	982047.9656				95412.45403				60306.3317	60306.3317			
D-Galactose	6374801.778			156406.4091		1737018.902	800493.3561	1432489.841	1116491.599	222796.8555			990900.4319		
Myo-Inositol	269628304.9	135993296.2	2358287.439	33391.31818	2626638.464	6334581.729	163831.8529	552956.7093	358394.2811	163199.7199			32406188.83	1428086.717	1589219.155
D-Ribose	4956714.856			139308.7955		400883.9878				4618616.888	11666172.71	11666172.71			1963440.106
D-deoxyribose						347778.4045		248906.5911				187244.8064	1047844.261		199393.0138
GLUTAMINIC ACID							186337.3638	3256712.792	1721525.078				413920.959		
D-Glucose	277287927.4	288918397.3	300548867.3		6018231.148		775598.3288	4904167.529	2839882.929	19947505.09	1796684.391	1796684.391	8538346.958		57010149.12
Pentadecanoic acid	20837919.64	13237890.28	5637860.912	264213.3636	642911.7276		202156.4253	388870.0879	295513.2566				420384.4101		
Hexadecanoic acid	301627500	185224076.5	68820653.01	1791062.273	3937129.713	362020.051	1666370.763	3787840.843	2727105.803		1125114.228	1125114.228	1565815.275	1942517.808	543347.729
Heptadecanoic acid	37223299.98	21953919.28	6684538.585	152350.6364	264966.6676		70502.46591							376406.5278	
Octadecanedioic acid	2053103.48														
Trehalose	958735.6435	775020.3869	591305.1302	38703.63636				92869.7567							52608.10371
Squalene	45259037.14	31220800.03	17182562.92	286762.2045		1098571.692	703701.9887	2653553.686	1678627.837						
EPA	2627189.064			86703.29545								1211891.901			
DHA	11091028.28	13076398.77	15061769.27	1347952.932	6243739.346		3257139.363	5133315.206	4195227.284				2263074.384		2382764.5
5-Methyluridine	3052333.584	3946286.664	4840239.744	133721.7273	271684.5144			814396.9788					668693.144		127251.36
5,6-Dimethyluracil			545211.8323	37257.93182		106736.3296							59962.38155		
Butanoic acid			87497493.23	1794506.023	3536288.294	1047614.823	1203444.52	1822845.434	1513144.977	46711.83922			2422315.231		
Cholesterol	45550968.73	32064163.51	18577358.29	227284.7727	889077.5314	4224346.723	874390.2302	4233996.068	2554193.149	370912.8392	579801.4118	579801.4118	940036.6743	4089498.473	1104113.245
D-Lactic acid	7833258.945	5541778.44	3250297.934	64358.20455	83365.48271		90229.32285	97872.20111	94050.76198				13065032.46		168262.5451
9-Octadecanoic acid			4073901.914	129080.6591				764037.1627							
Octadecanoic acid			97560604.75	1874310.114	1919258.233		348116.6921	241456.6334	294786.6628				729412.8165		
Eicosanoic acid			822314.1801												
Stigmasterol			57885259.73	75159.72727		1278792.157	283891.9523	636715.5227	460303.7375					1578315.667	411672.8714
D-Allose			96791100.9			3702039.66				13584990.21	19343870.32	19343870.32	3446879.167		
O,O''-Trimethylsilyl-O'-stearoylglycerol	6939435.106	3649640.609	359846.1121	28789.75				155921.4314							265422.5591
D-Erythrose			1355214.432	145481.4091				685459.982			1780625.493	280686.1978	1472989.752		800857.4521
Citric acid				96215.63636	75126.21706		322692.4167	768546.7203	545619.5685						
D-Tagatofuranose				268494.1364	361026.0117					482190.9556					3398727.791
D-Mannose					1029219.836	2298475.107	979972.531			4652816.492				206569.6525	2195488.55
Ribitol								221240.1951		109977.583	284992.7418	284992.7418			431310.071
D-sorbitol						928639.1479				3072077.617	404463.6062	404463.6062			1906660.183
Fumaric acid										295922.5371					163445.9093
XYLITOL								70725.78407		247170.5839					306353.1604
D-Arabinose			1479964.419	2343427.659	6356361.377								5507166.859	252707.5738	396261.3595
Docosanoic acid															4687210.421
D-Fructose										88679.62648					130574.7131
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	HMDB	PubChem	KEGG	SMILES	Comment
L-Alanine	HMDB0000161	5950	C00041	C[C@@H](C(=O)O)N	1
L-Glycine	HMDB0000123	750	C00037	C(C(=O)O)N	1
L-Valine	HMDB0000883	6287	C00183	CC(C)[C@@H](C(=O)O)N	1
L-Leucine	HMDB0000687	6106	C00123	CC(C)C[C@@H](C(=O)O)N	1
L-Proline	HMDB0000162	145742	C00148	C1C[C@H](NC1)C(=O)O	1
L-Isoleucine	HMDB0000172	6306	C00407	CC[C@H](C)[C@@H](C(=O)O)N	1
L-Serine	HMDB0000187	5951	C00065	C([C@@H](C(=O)O)N)O	1
L-Threonine	HMDB0000167	6288	C00188	C[C@H]([C@@H](C(=O)O)N)O	1
L-Methionine	HMDB0000696	6137	C00073	CSCC[C@@H](C(=O)O)N	1
L-Phenylalanine	HMDB0000159	6140	C00079	C1=CC=C(C=C1)C[C@@H](C(=O)O)N	1
L-Aspartic acid	HMDB0000191	5960	C00049	C([C@@H](C(=O)O)N)C(=O)O	1
L-Tyrosine	HMDB0000158	6057	C00082	C1=CC(=CC=C1C[C@@H](C(=O)O)N)O	1
L-Tryptophan	HMDB0000929	6305	C00078	C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)N	1
GABA	HMDB0000112	223130	C00334	C(CC(=O)O)CN	1
Propanoic acid	HMDB0000237	1032	C00163	CCC(=O)O	1
Acetic acid	HMDB0000042	176	C00033	CC(=O)O	1
Succinic acid	HMDB0000254	1110	C00042	C(CC(=O)O)C(=O)O	1
Malic acid	HMDB0000744	525	C03668	C(C(C(=O)O)O)C(=O)O	1
Glutamic acid	HMDB0000148	33032	C00025	C(CC(=O)O)[C@@H](C(=O)O)N	1
Phosphoric acid	HMDB0002142	57424078	C00009	OP(=O)(O)O	1
Glycerol	HMDB0000131	753	C00116	C(C(CO)O)O	1
Propanedioic acid	HMDB0000691	867	C04025	C(C(=O)O)C(=O)O	1
D-Galactose	HMDB0000143	439357	C00984	C([C@@H]1[C@@H]([C@@H]([C@H]([C@H](O1)O)O)O)O)O	1
Myo-Inositol	HMDB0000211	NA	C00137	O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O	1
D-Ribose	HMDB0000283	5779	C00121	C([C@@H]1[C@H]([C@H](C(O1)O)O)O)O	1
D-deoxyribose	HMDB0003224	22833604	C01801	C1[C@H]([C@@H](O[C@@H]1O)CO)O	1
GLUTAMINIC ACID	HMDB0000148	33032	C00025	C(CC(=O)O)[C@@H](C(=O)O)N	1
D-Glucose	HMDB0000122	5793	C00221	C([C@@H]1[C@H]([C@@H]([C@H](C(O1)O)O)O)O)O	1
Pentadecanoic acid	HMDB0000826	13849	C16537	CCCCCCCCCCCCCCC(=O)O	1
Hexadecanoic acid	HMDB0000220	985	C00249	CCCCCCCCCCCCCCCC(=O)O	1
Heptadecanoic acid	HMDB0002259	10465	NA	CCCCCCCCCCCCCCCCC(=O)O	1
Octadecanedioic acid	HMDB0000782	70095	NA	C(CCCCCCCCC(=O)O)CCCCCCCC(=O)O	1
Trehalose	HMDB0000975	7427	C01083	C([C@@H]1[C@H]([C@@H]([C@H]([C@H](O1)O[C@@H]2[C@@H]([C@H]([C@@H]([C@H](O2)CO)O)O)O)O)O)O)O	1
Squalene	HMDB0000256	11975273	C00751	CC(=CCC/C(=C/CC/C(=C/CC/C=C(C)/CC/C=C(/C)CCC=C(C)C)/C)/C)C	1
EPA	HMDB0001999	446284	C06428	CC/C=CC/C=CC/C=CC/C=CC/C=CCCCC(=O)O	1
DHA	HMDB0002183	445580	C06429	CC/C=CC/C=CC/C=CC/C=CC/C=CC/C=CCCC(=O)O	1
5-Methyluridine	HMDB0000884	445408	NA	CC1=CN(C(=O)NC1=O)[C@H]2[C@@H]([C@@H]([C@H](O2)CO)O)O	1
5,6-Dimethyluracil	NA	NA	NA	NA	0
Butanoic acid	HMDB0000039	264	C00246	CCCC(=O)O	1
Cholesterol	HMDB0000067	5997	C00187	C[C@H](CCCC(C)C)[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2CC=C4[C@@]3(CCC(C4)O)C)C	1
D-Lactic acid	HMDB0001311	61503	C00256	C[C@H](C(=O)O)O	1
9-Octadecanoic acid	NA	NA	NA	NA	0
Octadecanoic acid	HMDB0000827	5281	C01530	CCCCCCCCCCCCCCCCCC(=O)O	1
Eicosanoic acid	HMDB0002212	10467	C06425	CCCCCCCCCCCCCCCCCCCC(=O)O	1
Stigmasterol	HMDB0000937	5281330	C05442	CC[C@H](/C=C/[C@@H](C)[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2CC=C4[C@@]3(CC[C@@H](C4)O)C)C)C(C)C	1
D-Allose	HMDB0001151	12285879	C01487	C([C@H]1[C@@H]([C@@H]([C@@H](C(O1)O)O)O)O)O	1
O,O''-Trimethylsilyl-O'-stearoylglycerol	NA	NA	NA	NA	0
D-Erythrose	HMDB0002649	5460672	C01796	C1[C@H]([C@H](C(O1)O)O)O	1
Citric acid	HMDB0000094	311	C00158	C(C(=O)O)C(CC(=O)O)(C(=O)O)O	1
D-Tagatofuranose	NA	NA	NA	NA	0
D-Mannose	HMDB0000169	18950	C00936	C([C@@H]1[C@H]([C@@H]([C@@H](C(O1)O)O)O)O)O	1
Ribitol	HMDB0000508	NA	C00474	OC[C@H](O)[C@H](O)[C@H](O)CO	1
D-sorbitol	HMDB0000247	5780	C00794	C([C@H]([C@H]([C@@H]([C@H](CO)O)O)O)O)O	1
Fumaric acid	HMDB0000134	444972	C00122	C(=C/C(=O)O)C(=O)O	1
XYLITOL	HMDB0002917	6912	C00379	C([C@H](C([C@H](CO)O)O)O)O	1
D-Arabinose	HMDB0029942	66308	NA	C([C@H]([C@H]([C@@H](C=O)O)O)O)O	1
Docosanoic acid	HMDB0000944	8215	C08281	CCCCCCCCCCCCCCCCCCCCCC(=O)O	1
D-Fructose	HMDB0000660	439709	C02336	C([C@@H]1[C@H]([C@@H]([C@](O1)(CO)O)O)O)O	1
METABOLITES_END
#END