#METABOLOMICS WORKBENCH dassmetabolomics_20250327_144650 DATATRACK_ID:5799 STUDY_ID:ST003828 ANALYSIS_ID:AN006288 PROJECT_ID:PR002391
VERSION             	1
CREATED_ON             	March 31, 2025, 1:39 pm
#PROJECT
PR:PROJECT_TITLE                 	An integrated analysis of behavioral and metabolic modulations under a
PR:PROJECT_TITLE                 	chronobiotic treatment in Drosophila melanogaster
PR:PROJECT_TYPE                  	MS based Metabolomics analysis
PR:PROJECT_SUMMARY               	Drosophila melanogaster, with its conserved molecular clocks and accessible
PR:PROJECT_SUMMARY               	assays, serves as an ideal model system for studying circadian rhythms and
PR:PROJECT_SUMMARY               	sleep. Using an integrated approach combining high-throughput locomotor activity
PR:PROJECT_SUMMARY               	monitoring and untargeted metabolomics, we analyzed the behavioral and metabolic
PR:PROJECT_SUMMARY               	effects of a chronobiotic melatonin. The behavioral activity of flies was
PR:PROJECT_SUMMARY               	recorded using an infrared-based monitoring device followed by data analysis
PR:PROJECT_SUMMARY               	with open-source data packages, ShinyR-DAM and VANESSA. We found that 1mM and
PR:PROJECT_SUMMARY               	4mM melatonin doses significantly increased Drosophila locomotor activity.
PR:PROJECT_SUMMARY               	Melatonin at high concentrations (4mM) exhibited protective effects by reducing
PR:PROJECT_SUMMARY               	mortality in Drosophila. Despite these changes, melatonin preserved the flies;
PR:PROJECT_SUMMARY               	endogenous bimodal activity pattern, maintaining circadian alignment. High-
PR:PROJECT_SUMMARY               	performance LC-MS metabolomics identified differentially abundant metabolites
PR:PROJECT_SUMMARY               	under drug treatment. LC-MS-based metabolomics identified 20 biologically
PR:PROJECT_SUMMARY               	relevant metabolites altered by melatonin, including key perturbations in
PR:PROJECT_SUMMARY               	arginine biosynthesis, alanine/aspartate/glutamate metabolism, and pyrimidine
PR:PROJECT_SUMMARY               	pathways. Notably, melatonin upregulated glutamine, a potential indicator of
PR:PROJECT_SUMMARY               	enhanced neurotransmitter synthesis and broadly modulated amino acid and
PR:PROJECT_SUMMARY               	nucleotide metabolism, suggesting dual roles in neuroprotection and energy
PR:PROJECT_SUMMARY               	homeostasis. This high-throughput approach uncovers melatonin- induced
PR:PROJECT_SUMMARY               	behavioral and metabolic perturbations, revealing how melatonin modulates
PR:PROJECT_SUMMARY               	locomotor activity, survival, and circadian integrity through specific
PR:PROJECT_SUMMARY               	alterations in metabolism.
PR:INSTITUTE                     	Indian Institute of Technology Hyderabad
PR:DEPARTMENT                    	Biotechnology
PR:LABORATORY                    	Circadian Rhythms and Disease Biology Laboratory
PR:LAST_NAME                     	Das
PR:FIRST_NAME                    	Sandip
PR:ADDRESS                       	NH-9,KANDI, sangareddy, telengana, 502285, India
PR:EMAIL                         	bo21resch11009@iith.ac.in
PR:PHONE                         	6290857805
#STUDY
ST:STUDY_TITLE                   	An integrated analysis of behavioral and metabolic modulations under a
ST:STUDY_TITLE                   	chronobiotic treatment in Drosophila melanogaster
ST:STUDY_SUMMARY                 	Drosophila melanogaster, with its conserved molecular clocks and accessible
ST:STUDY_SUMMARY                 	assays, serves as an ideal model system for studying circadian rhythms and
ST:STUDY_SUMMARY                 	sleep. Using an integrated approach combining high-throughput locomotor activity
ST:STUDY_SUMMARY                 	monitoring and untargeted metabolomics, we analyzed the behavioral and metabolic
ST:STUDY_SUMMARY                 	effects of a chronobiotic melatonin. The behavioral activity of flies was
ST:STUDY_SUMMARY                 	recorded using an infrared-based monitoring device followed by data analysis
ST:STUDY_SUMMARY                 	with open-source data packages, ShinyR-DAM and VANESSA. We found that 1mM and
ST:STUDY_SUMMARY                 	4mM melatonin doses significantly increased Drosophila locomotor activity.
ST:STUDY_SUMMARY                 	Melatonin at high concentrations (4mM) exhibited protective effects by reducing
ST:STUDY_SUMMARY                 	mortality in Drosophila. Despite these changes, melatonin preserved the flies;
ST:STUDY_SUMMARY                 	endogenous bimodal activity pattern, maintaining circadian alignment. High-
ST:STUDY_SUMMARY                 	performance LC-MS metabolomics identified differentially abundant metabolites
ST:STUDY_SUMMARY                 	under drug treatment. LC-MS-based metabolomics identified 20 biologically
ST:STUDY_SUMMARY                 	relevant metabolites altered by melatonin, including key perturbations in
ST:STUDY_SUMMARY                 	arginine biosynthesis, alanine/aspartate/glutamate metabolism, and pyrimidine
ST:STUDY_SUMMARY                 	pathways. Notably, melatonin upregulated glutamine, a potential indicator of
ST:STUDY_SUMMARY                 	enhanced neurotransmitter synthesis and broadly modulated amino acid and
ST:STUDY_SUMMARY                 	nucleotide metabolism, suggesting dual roles in neuroprotection and energy
ST:STUDY_SUMMARY                 	homeostasis. This high-throughput approach uncovers melatonin- induced
ST:STUDY_SUMMARY                 	behavioral and metabolic perturbations, revealing how melatonin modulates
ST:STUDY_SUMMARY                 	locomotor activity, survival, and circadian integrity through specific
ST:STUDY_SUMMARY                 	alterations in metabolism.
ST:INSTITUTE                     	Indian Institute of Technology Hyderabad
ST:DEPARTMENT                    	Biotechnology
ST:LABORATORY                    	Circadian Rhythms and Disease Biology Laboratory
ST:LAST_NAME                     	Das
ST:FIRST_NAME                    	Sandip
ST:ADDRESS                       	NH-9,KANDI
ST:EMAIL                         	bo21resch11009@iith.ac.in
ST:PHONE                         	6290857805
#SUBJECT
SU:SUBJECT_TYPE                  	Invertebrate
SU:SUBJECT_SPECIES               	Drosophila melanogaster
SU:TAXONOMY_ID                   	7227
SU:GENOTYPE_STRAIN               	Canton-S
SU:AGE_OR_AGE_RANGE              	3-4 day
SU:GENDER                        	Male
#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           	-	Sa_1	Sample source:Fly head | Treatment:Control	RAW_FILE_NAME(Raw file name)=Control R1-r001.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_2	Sample source:Fly head | Treatment:Control	RAW_FILE_NAME(Raw file name)=Control R1-r002.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_3	Sample source:Fly head | Treatment:Control	RAW_FILE_NAME(Raw file name)=Control R1-r003.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_4	Sample source:Fly head | Treatment:Control	RAW_FILE_NAME(Raw file name)=Control R2-r001.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_5	Sample source:Fly head | Treatment:Control	RAW_FILE_NAME(Raw file name)=Control R2-r002.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_6	Sample source:Fly head | Treatment:Control	RAW_FILE_NAME(Raw file name)=Control R2-r003.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_7	Sample source:Fly head | Treatment:Control	RAW_FILE_NAME(Raw file name)=Control R3-r001.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_8	Sample source:Fly head | Treatment:Control	RAW_FILE_NAME(Raw file name)=Control R3-r002.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_9	Sample source:Fly head | Treatment:Control	RAW_FILE_NAME(Raw file name)=Control R3-r003.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_10	Sample source:Fly head | Treatment:Melatonin_Treated	RAW_FILE_NAME(Raw file name)=Mel R1-r001.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_11	Sample source:Fly head | Treatment:Melatonin_Treated	RAW_FILE_NAME(Raw file name)=Mel R1-r002.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_12	Sample source:Fly head | Treatment:Melatonin_Treated	RAW_FILE_NAME(Raw file name)=Mel R1-r003.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_13	Sample source:Fly head | Treatment:Melatonin_Treated	RAW_FILE_NAME(Raw file name)=Mel R2-r001.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_14	Sample source:Fly head | Treatment:Melatonin_Treated	RAW_FILE_NAME(Raw file name)=Mel R2-r002.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_15	Sample source:Fly head | Treatment:Melatonin_Treated	RAW_FILE_NAME(Raw file name)=Mel R2-r003.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_16	Sample source:Fly head | Treatment:Melatonin_Treated	RAW_FILE_NAME(Raw file name)=Mel R3-r001.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_17	Sample source:Fly head | Treatment:Melatonin_Treated	RAW_FILE_NAME(Raw file name)=Mel R3-r002.d
SUBJECT_SAMPLE_FACTORS           	-	Sa_18	Sample source:Fly head | Treatment:Melatonin_Treated	RAW_FILE_NAME(Raw file name)=Mel R3-r003.d
#COLLECTION
CO:COLLECTION_SUMMARY            	Melatonin 48-h treated (2 circadian cycles) and equivalent ethanol-treated flies
CO:COLLECTION_SUMMARY            	were considered for metabolomics. 24 flies from each vial were considered one
CO:COLLECTION_SUMMARY            	biological sample. Three sets of these samples were created for melatonin and
CO:COLLECTION_SUMMARY            	vehicle (biological replicates), resulting in six samples. Flies were
CO:COLLECTION_SUMMARY            	snap-frozen using liquid nitrogen and stored frozen at −80 °C until heads
CO:COLLECTION_SUMMARY            	were separated and used for metabolite extractionmetabolite extraction.
CO:SAMPLE_TYPE                   	Fly Head
#TREATMENT
TR:TREATMENT_SUMMARY             	3 to 4-day-old adult imago male flies were supplemented with fresh solutions of
TR:TREATMENT_SUMMARY             	4 mM Melatonin crystalline (the stock concentration of the drug was dissolved in
TR:TREATMENT_SUMMARY             	95% ethanol) in the sucrose-agar media. Equivalent volumes and concentrations of
TR:TREATMENT_SUMMARY             	the vehicle were added to the sucrose-agar medium for vehicle treatments.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Heads were separated and used for metabolite extraction by adding a mixture of
SP:SAMPLEPREP_SUMMARY            	cold 120 μl methanol: chloroform (2:1). The samples were homogenized for 3
SP:SAMPLEPREP_SUMMARY            	cycles on ice (3 mins on, 3 mins off). Mild sonication was applied for 15 min
SP:SAMPLEPREP_SUMMARY            	(30s on, 30s off; high power) using a Bioruptor Standard (Diagenode) instrument.
SP:SAMPLEPREP_SUMMARY            	A 200 μl of an additional solvent of water and chloroform (1:1) was added to
SP:SAMPLEPREP_SUMMARY            	the homogenate. After centrifugation (18,000 g for 7 minutes at 4°C), 100 μl
SP:SAMPLEPREP_SUMMARY            	of the aqueous layer was collected in a fresh microcentrifuge tube. The
SP:SAMPLEPREP_SUMMARY            	separated aqueous layer was dried down under a vacuum at 1500 rpm. The dried
SP:SAMPLEPREP_SUMMARY            	pellets were re-suspended in a 100 μl mixture of water and acetonitrile (1:1)
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Agilent 6550
CH:COLUMN_NAME                   	Agilent InfinityLab Poroshell 120 HILIC-Z (100 x 2.1mm, 2.7um)
CH:SOLVENT_A                     	100% water; 10 mM of ammonium acetate
CH:SOLVENT_B                     	85% acetonitrile/15% water; 10 mM of ammonium acetate
CH:FLOW_GRADIENT                 	0 min, 96 % B; 0-2 min 96 % B, 2-5.5 min, 96-88% B; 5.5-8.5 min, 88% B; 8.5-9
CH:FLOW_GRADIENT                 	min, 88-86% B; 9-14 min, 86% B; 14-17 min, 86-82% B; 17-23 min, 82-65% B; 23-24
CH:FLOW_GRADIENT                 	min, 65% B; 24-24.5 min, 65-96% B; 24.5-26 min, 96% B.
CH:FLOW_RATE                     	0.25 mL/min
CH:COLUMN_TEMPERATURE            	50
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6550 QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	The data acquisition process used Agilent MassHunter Workstation Data
MS:MS_COMMENTS                   	Acquisition software (version B.05.01). Raw data files in Agilent '.d' format
MS:MS_COMMENTS                   	were processed using MassHunter Profinder software. The software utilized a
MS:MS_COMMENTS                   	batch molecular feature extraction algorithm with parameters set at a threshold
MS:MS_COMMENTS                   	of 1000 counts and a maximum charge state of 2. In negative ionization mode,
MS:MS_COMMENTS                   	adduct formation of −H and +HCOO was utilized to identify features of the same
MS:MS_COMMENTS                   	molecule. Feature alignment was performed with a tolerance window of 0.30 min
MS:MS_COMMENTS                   	and mass accuracy of 5 ppm for retention times and m/z values across all data
MS:MS_COMMENTS                   	files. Subsequently, feature-extracted data files were exported from Profinder
MS:MS_COMMENTS                   	as '.CEF' files for each profiling experiment.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Abundance
MS_METABOLITE_DATA_START
Samples	Sa_1	Sa_2	Sa_3	Sa_4	Sa_5	Sa_6	Sa_7	Sa_8	Sa_9	Sa_10	Sa_11	Sa_12	Sa_13	Sa_14	Sa_15	Sa_16	Sa_17	Sa_18
Factors	Sample source:Fly head | Treatment:Control	Sample source:Fly head | Treatment:Control	Sample source:Fly head | Treatment:Control	Sample source:Fly head | Treatment:Control	Sample source:Fly head | Treatment:Control	Sample source:Fly head | Treatment:Control	Sample source:Fly head | Treatment:Control	Sample source:Fly head | Treatment:Control	Sample source:Fly head | Treatment:Control	Sample source:Fly head | Treatment:Melatonin_Treated	Sample source:Fly head | Treatment:Melatonin_Treated	Sample source:Fly head | Treatment:Melatonin_Treated	Sample source:Fly head | Treatment:Melatonin_Treated	Sample source:Fly head | Treatment:Melatonin_Treated	Sample source:Fly head | Treatment:Melatonin_Treated	Sample source:Fly head | Treatment:Melatonin_Treated	Sample source:Fly head | Treatment:Melatonin_Treated	Sample source:Fly head | Treatment:Melatonin_Treated
L-Glutamine	3.06E+07	3.03E+07	3.08E+07	3.56E+07	3.60E+07	3.51E+07	3.21E+07	3.22E+07	3.27E+07	7.39E+07	7.40E+07	7.44E+07	1.32E+08	1.33E+08	1.34E+08	1.16E+08	1.16E+08	1.16E+08
L-Aspartic Acid	7708323	8805905	9130127	5750704	6498195	7391742	1.42E+07	1.46E+07	1.56E+07	3.99E+07	4.17E+07	4.13E+07	3.62E+07	3.62E+07	3.68E+07	4.20E+07	4.23E+07	4.15E+07
dCYD / 2'-Deoxycytidine	4543926	4590664	4578390	5888500	5997384	5899234	6204863	3032778	4715418	1.37E+07	1.42E+07	1.43E+07	3.48E+07	3.54E+07	3.53E+07	2.28E+07	2.31E+07	2.32E+07
L-Leucine	4214812	4046259	3947112	4448851	4573714	4593434	3079294	3186080	3075180	5009229	5021410	5072299	6638274	6820114	6857386	6463431	6553969	6602465
alpha-Ketoglutaric acid	9232473	9066872	8627568	4919321	4807061	4644822	4307944	4455076	4155723	1612855	1464320	1631843	1842753	1808703	1819487	2647885	2781773	2793617
2-keto-D-Gluconic acid	1.47E+07	1.40E+07	1.37E+07	8519294	8800107	8159694	4184815	4438108	4163173	3907582	3759208	3691701	4390360	4358333	4295692	3931474	3798232	3748100
Cytidine	630073	633226	685880	842296	1183910	863050	1717387	1630955	1647895	2664628	2867547	2796556	2489662	2563534	2554388	2223345	2295460	2338011
(±)5,6-DHET	2317318	2118699	2357874	2267678	2500694	2402617	2961710	2912230	2753730	6831330	6845405	6934962	6001398	6057582	5988073	5454565	6421447	6141894
Galactosylglycerol	1345181	1342574	1307109	1844134	1852821	632868	2207644	2271317	2254294	9595168	2909003	5160789	2.52E+07	2.62E+07	2.43E+07	1.70E+07	1.74E+07	1.79E+07
L-Methionine S-oxide	1.50E+07	1.51E+07	1.56E+07	1.21E+07	1.15E+07	1.16E+07	1.62E+07	1.63E+07	1.59E+07	9103837	9088802	8924580	8313760	8365988	8450416	5385872	5662396	5445896
Phytyl diphosphate	469216	471824	480609	314280	544833	453431	551144	540823	572133	1758456	1687782	1585607	1441875	1378117	1404456	1419428	1293202	1334074
PE(18:0/15:0)	260458	265471	274004	155347	173057	166573	371451	342494	308700	129961	126458	121089	167912	163763	172773	170028	167957	155567
PE(20:3(8Z,11Z,14Z)/14:0)	255811	249365	233415	271334	276524	247984	201530	183729	169820	229100	226499	211858	167275	164070	161233	154735	162530	142614
4-Imidazolone-5-propionic acid	2840771	3049291	3157921	4063112	4194662	4125666	3042569	3087345	3025538	3480856	3415014	3477387	7221276	7291470	7257213	5870675	5817083	5892119
Stachyose	4.41E+07	4.54E+07	4.52E+07	6.96E+07	6.89E+07	6.87E+07	5.79E+07	5.76E+07	5.76E+07	2.35E+07	2.30E+07	2.33E+07	2.61E+07	2.61E+07	2.59E+07	3.70E+07	3.59E+07	3.60E+07
Uric acid	3.73E+08	3.67E+08	3.45E+08	3.58E+08	3.57E+08	3.50E+08	3.54E+08	3.29E+08	3.31E+08	4.23E+07	3.77E+07	3.68E+07	1.94E+08	1.87E+08	1.73E+08	1.05E+08	1.06E+08	1.04E+08
2-Ketobutyrate	7222175	7451840	7007225	3831284	3525941	3575920	3160297	3268779	3189054	1425599	1358878	1409594	1626052	1670548	1691285	2312289	2381356	2456497
Raffinose	7039931	6982724	7104861	5235875	6314513	5901085	5137980	4992954	5047411	1.04E+07	1.03E+07	1.04E+07	6781251	7216341	7026507	6847543	6803057	6613585
D-Glycerol 1-phosphate	1.59E+08	1.58E+08	1.55E+08	4.42E+08	4.08E+08	4.21E+08	6.34E+07	6.63E+07	6.94E+07	4.67E+07	5.26E+07	5.23E+07	3.88E+07	3.20E+07	3.10E+07	1.32E+08	1.35E+08	1.32E+08
Histamine	4412655	4326892	4240004	1475506	4015552	4064097	2241886	2162769	3407905	4528826	4145555	2636779	6361725	6394215	4876860	5661296	5627153	5630971
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	retention index	Mass	KEGG ID
L-Glutamine	5.9775004	146.0692	C00064
dCYD / 2'-Deoxycytidine	5.887501	273.0936	C00881
L-Leucine	2.7269995	131.0944	C00123
4-Imidazolone-5-propionic acid	5.716499	202.0564	C03680
Raffinose	7.2865	504.1683	C00492
Uric acid	3.0510004	168.0271	C00366
2-keto-D-Gluconic acid	5.813001	194.0428	C06473
D-Glycerol 1-phosphate	9.263498	172.0144	C00093
Cytidine	5.8444996	243.0831	C00475
alpha-Ketoglutaric acid	7.1495013	146.0212	C00026
Histamine	7.673	111.079	C00388
Galactosylglycerol	5.948499	314.1202	C05401
Stachyose	8.637999	666.2225	C01613
2-Ketobutyrate	7.1540017	102.0314	C00109
L-Aspartic Acid	2.9004996	179.0439	C00049
(±)5,6-DHET	5.5865	338.2428	C14772
L-Methionine S-oxide	6.3049984	165.0455	C02989
Phytyl diphosphate	5.4755	502.2487	C05427
PE(18:0/15:0)	2.2714999	751.5344	C00350
PE(20:3(8Z,11Z,14Z)/14:0)	2.2245002	759.5024	C00350
METABOLITES_END
#END