#METABOLOMICS WORKBENCH carolinejohnson2016_20251216_062817 DATATRACK_ID:6898 STUDY_ID:ST004470 ANALYSIS_ID:AN007494 PROJECT_ID:PR002823
VERSION             	1
CREATED_ON             	December 18, 2025, 2:11 pm
#PROJECT
PR:PROJECT_TITLE                 	Sex-specific effects of exogenous asparagine on colorectal tumor growth,
PR:PROJECT_TITLE                 	17β-estradiol levels, and aromatase in mice
PR:PROJECT_SUMMARY               	The study aims to understand the relationship between exogenous asparagine
PR:PROJECT_SUMMARY               	supplementation and 17β-estradiol levels and to elucidate mechanisms underlying
PR:PROJECT_SUMMARY               	sex-dependent signaling during CRC development. We administered asparagine
PR:PROJECT_SUMMARY               	intraperitoneally to tumor-bearing male and female immunodeficient Rag2/Il2RG
PR:PROJECT_SUMMARY               	(R2G2) mice. We performed untargeted metabolomics and found that intraperitoneal
PR:PROJECT_SUMMARY               	asparagine treatment induced sex-dependent intra-tumoral metabolic changes to
PR:PROJECT_SUMMARY               	asparagine, aspartate, glutamine and glutamate levels. To confirm whether
PR:PROJECT_SUMMARY               	asparagine supplementation alters de novo asparagine synthesis in a sex-specific
PR:PROJECT_SUMMARY               	manner, we performed untargeted metabolomics on the tumor xenografts from the
PR:PROJECT_SUMMARY               	R2G2 mice. We examined abundances of metabolites that are involved in the
PR:PROJECT_SUMMARY               	asparagine metabolism pathway including aspartate and glutamine (substrates) and
PR:PROJECT_SUMMARY               	asparagine and glutamate (products). Samples were analyzed in hydrophilic
PR:PROJECT_SUMMARY               	interaction chromatography (HILIC) and reverse phase (RPLC) mass spectrometry
PR:PROJECT_SUMMARY               	(MS) modes. The mass spectrometry data was acquired using a UPLC system (H-Class
PR:PROJECT_SUMMARY               	ACQUITY, Waters Corporation, MA, United States) coupled to a quadrupole time-of
PR:PROJECT_SUMMARY               	flight (QTOF) mass spectrometer (Xevo G2-XS QTOF, Waters Corporation, MA, United
PR:PROJECT_SUMMARY               	States).
PR:INSTITUTE                     	Yale University
PR:DEPARTMENT                    	Environmental Health Sciences
PR:LABORATORY                    	Caroline Johnson
PR:LAST_NAME                     	Johnson
PR:FIRST_NAME                    	Caroline
PR:ADDRESS                       	60 College Street, LEPH, Room 509
PR:EMAIL                         	caroline.johnson@yale.edu
PR:PHONE                         	2037852882
PR:FUNDING_SOURCE                	R01 CA289440/CA/NCI NIH HHS/United States
PR:PUBLICATIONS                  	https://pubmed.ncbi.nlm.nih.gov/40228761/
PR:CONTRIBUTORS                  	Oladimeji Aladelokun, Katherine Benitez, Yuying Wang, Abhishek Jain, Domenica
PR:CONTRIBUTORS                  	Berardi, Georgio Maroun, Xinyi Shen, Jatin Roper, Joanna Gibson, Kaelyn
PR:CONTRIBUTORS                  	Sumigray, Sajid A Khan, Caroline H Johnson
#STUDY
ST:STUDY_TITLE                   	Sex-specific effects of exogenous asparagine on colorectal tumor growth,
ST:STUDY_TITLE                   	17β-estradiol levels, and aromatase in mice
ST:STUDY_SUMMARY                 	The study aims to understand the relationship between exogenous asparagine
ST:STUDY_SUMMARY                 	supplementation and 17β-estradiol levels and to elucidate mechanisms underlying
ST:STUDY_SUMMARY                 	sex-dependent signaling during CRC development. We administered asparagine
ST:STUDY_SUMMARY                 	intraperitoneally to tumor-bearing male and female immunodeficient Rag2/Il2RG
ST:STUDY_SUMMARY                 	(R2G2) mice. We performed untargeted metabolomics and found that intraperitoneal
ST:STUDY_SUMMARY                 	asparagine treatment induced sex-dependent intra-tumoral metabolic changes to
ST:STUDY_SUMMARY                 	asparagine, aspartate, glutamine and glutamate levels.
ST:INSTITUTE                     	Yale University
ST:LAST_NAME                     	Johnson
ST:FIRST_NAME                    	Caroline
ST:ADDRESS                       	60 College Street, LEPH, Room 509
ST:EMAIL                         	caroline.johnson@yale.edu
ST:PHONE                         	2037852882
ST:NUM_GROUPS                    	6
ST:TOTAL_SUBJECTS                	29
ST:NUM_MALES                     	15
ST:NUM_FEMALES                   	14
ST:PUBLICATIONS                  	https://pubmed.ncbi.nlm.nih.gov/40228761/
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
SU:GENOTYPE_STRAIN               	Rag2/IL2RG
SU:AGE_OR_AGE_RANGE              	4-6 weeks
SU:WEIGHT_OR_WEIGHT_RANGE        	25 g
SU:HEIGHT_OR_HEIGHT_RANGE        	N/A
SU:GENDER                        	Male and female
SU:ANIMAL_ANIMAL_SUPPLIER        	Inotiv
SU:ANIMAL_HOUSING                	Yale School of Medicine SPF facility
SU:ANIMAL_LIGHT_CYCLE            	12-hour light and dark
SU:ANIMAL_FEED                   	Standard rodent chow diet; Teklad #2018S
SU:ANIMAL_WATER                  	ad libitum
SU:ANIMAL_INCLUSION_CRITERIA     	N/A
SU:SPECIES_GROUP                 	N/A
#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           	-	601	Sample source:Tumor xenograft_Female R2G2 | Factor:Control	Sex=Female; RAW_FILE_NAME(Raw file name)=601.mzML
SUBJECT_SAMPLE_FACTORS           	-	602	Sample source:Tumor xenograft_Female R2G2 | Factor:Control	Sex=Female; RAW_FILE_NAME(Raw file name)=602.mzML
SUBJECT_SAMPLE_FACTORS           	-	604	Sample source:Tumor xenograft_Female R2G2 | Factor:Control	Sex=Female; RAW_FILE_NAME(Raw file name)=604.mzML
SUBJECT_SAMPLE_FACTORS           	-	605	Sample source:Tumor xenograft_Female R2G2 | Factor:Control	Sex=Female; RAW_FILE_NAME(Raw file name)=605.mzML
SUBJECT_SAMPLE_FACTORS           	-	606	Sample source:Tumor xenograft_Female R2G2 | Factor:1 mg/kg asparagine	Sex=Female; RAW_FILE_NAME(Raw file name)=606.mzML
SUBJECT_SAMPLE_FACTORS           	-	607	Sample source:Tumor xenograft_Female R2G2 | Factor:1 mg/kg asparagine	Sex=Female; RAW_FILE_NAME(Raw file name)=607.mzML
SUBJECT_SAMPLE_FACTORS           	-	608	Sample source:Tumor xenograft_Female R2G2 | Factor:1 mg/kg asparagine	Sex=Female; RAW_FILE_NAME(Raw file name)=608.mzML
SUBJECT_SAMPLE_FACTORS           	-	609	Sample source:Tumor xenograft_Female R2G2 | Factor:1 mg/kg asparagine	Sex=Female; RAW_FILE_NAME(Raw file name)=609.mzML
SUBJECT_SAMPLE_FACTORS           	-	610	Sample source:Tumor xenograft_Female R2G2 | Factor:1 mg/kg asparagine	Sex=Female; RAW_FILE_NAME(Raw file name)=610.mzML
SUBJECT_SAMPLE_FACTORS           	-	611	Sample source:Tumor xenograft_Female R2G2 | Factor:10 mg/kg asparagine	Sex=Female; RAW_FILE_NAME(Raw file name)=611.mzML
SUBJECT_SAMPLE_FACTORS           	-	612	Sample source:Tumor xenograft_Female R2G2 | Factor:10 mg/kg asparagine	Sex=Female; RAW_FILE_NAME(Raw file name)=612.mzML
SUBJECT_SAMPLE_FACTORS           	-	613	Sample source:Tumor xenograft_Female R2G2 | Factor:10 mg/kg asparagine	Sex=Female; RAW_FILE_NAME(Raw file name)=613.mzML
SUBJECT_SAMPLE_FACTORS           	-	614	Sample source:Tumor xenograft_Female R2G2 | Factor:10 mg/kg asparagine	Sex=Female; RAW_FILE_NAME(Raw file name)=614.mzML
SUBJECT_SAMPLE_FACTORS           	-	615	Sample source:Tumor xenograft_Female R2G2 | Factor:10 mg/kg asparagine	Sex=Female; RAW_FILE_NAME(Raw file name)=615.mzML
SUBJECT_SAMPLE_FACTORS           	-	616	Sample source:Tumor xenograft_Male R2G2 | Factor:Control	Sex=Male; RAW_FILE_NAME(Raw file name)=616.mzML
SUBJECT_SAMPLE_FACTORS           	-	617	Sample source:Tumor xenograft_Male R2G2 | Factor:Control	Sex=Male; RAW_FILE_NAME(Raw file name)=617.mzML
SUBJECT_SAMPLE_FACTORS           	-	618	Sample source:Tumor xenograft_Male R2G2 | Factor:Control	Sex=Male; RAW_FILE_NAME(Raw file name)=618.mzML
SUBJECT_SAMPLE_FACTORS           	-	619	Sample source:Tumor xenograft_Male R2G2 | Factor:Control	Sex=Male; RAW_FILE_NAME(Raw file name)=619.mzML
SUBJECT_SAMPLE_FACTORS           	-	620	Sample source:Tumor xenograft_Male R2G2 | Factor:Control	Sex=Male; RAW_FILE_NAME(Raw file name)=620.mzML
SUBJECT_SAMPLE_FACTORS           	-	621	Sample source:Tumor xenograft_Male R2G2 | Factor:1 mg/kg asparagine	Sex=Male; RAW_FILE_NAME(Raw file name)=621.mzML
SUBJECT_SAMPLE_FACTORS           	-	622	Sample source:Tumor xenograft_Male R2G2 | Factor:1 mg/kg asparagine	Sex=Male; RAW_FILE_NAME(Raw file name)=622.mzML
SUBJECT_SAMPLE_FACTORS           	-	623	Sample source:Tumor xenograft_Male R2G2 | Factor:1 mg/kg asparagine	Sex=Male; RAW_FILE_NAME(Raw file name)=623.mzML
SUBJECT_SAMPLE_FACTORS           	-	624	Sample source:Tumor xenograft_Male R2G2 | Factor:1 mg/kg asparagine	Sex=Male; RAW_FILE_NAME(Raw file name)=624.mzML
SUBJECT_SAMPLE_FACTORS           	-	625	Sample source:Tumor xenograft_Male R2G2 | Factor:1 mg/kg asparagine	Sex=Male; RAW_FILE_NAME(Raw file name)=625.mzML
SUBJECT_SAMPLE_FACTORS           	-	626	Sample source:Tumor xenograft_Male R2G2 | Factor:10 mg/kg asparagine	Sex=Male; RAW_FILE_NAME(Raw file name)=626.mzML
SUBJECT_SAMPLE_FACTORS           	-	627	Sample source:Tumor xenograft_Male R2G2 | Factor:10 mg/kg asparagine	Sex=Male; RAW_FILE_NAME(Raw file name)=627.mzML
SUBJECT_SAMPLE_FACTORS           	-	628	Sample source:Tumor xenograft_Male R2G2 | Factor:10 mg/kg asparagine	Sex=Male; RAW_FILE_NAME(Raw file name)=628.mzML
SUBJECT_SAMPLE_FACTORS           	-	629	Sample source:Tumor xenograft_Male R2G2 | Factor:10 mg/kg asparagine	Sex=Male; RAW_FILE_NAME(Raw file name)=629.mzML
SUBJECT_SAMPLE_FACTORS           	-	630	Sample source:Tumor xenograft_Male R2G2 | Factor:10 mg/kg asparagine	Sex=Male; RAW_FILE_NAME(Raw file name)=630.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	Tumor tissue was homogenized in UPLC-grade H2O using the Precellys Evolution
CO:COLLECTION_SUMMARY            	cryo-homogenizer (Bertin Corp. USA) and metabolites were extracted using
CO:COLLECTION_SUMMARY            	methanol:acetonitrile (1:1, v/v). For a comprehensive analysis of the
CO:COLLECTION_SUMMARY            	metabolome, untargeted metabolomics was conducted as described below. Briefly,
CO:COLLECTION_SUMMARY            	samples were analyzed in hydrophilic interaction chromatography (HILIC) mass
CO:COLLECTION_SUMMARY            	spectrometry (MS) mode. The mass spectrometry data was acquired using a UPLC
CO:COLLECTION_SUMMARY            	system (H-Class ACQUITY, Waters Corporation, MA, United States) coupled to a
CO:COLLECTION_SUMMARY            	quadrupole time-of flight (QTOF) mass spectrometer (Xevo G2-XS QTOF, Waters
CO:COLLECTION_SUMMARY            	Corporation, MA, United States). Waters ACQUITY UPLC BEH Amide column [particle
CO:COLLECTION_SUMMARY            	size: 1.7 μm; 100 mm (length) by 2.1 mm (internal diameter)] and a BEH C18
CO:COLLECTION_SUMMARY            	column [particle size: 1.7 μm; 100 mm (length) by 2.1 mm (internal diameter)]
CO:COLLECTION_SUMMARY            	were used for the UPLC-based separation of metabolites. For HILIC-MS run, the
CO:COLLECTION_SUMMARY            	mobile phase A was 25 mM ammonium hydroxide and 25 mM ammonium acetate in water
CO:COLLECTION_SUMMARY            	while mobile phase B was acetonitrile. For RPLC-MS run, mobile phase A was 0.1%
CO:COLLECTION_SUMMARY            	formic acid in water, while mobile phase B was 0.1% formic acid in acetonitrile.
CO:COLLECTION_SUMMARY            	Pooled samples were analyzed at specified injections interval during the UPLC-MS
CO:COLLECTION_SUMMARY            	analysis which allowed monitoring of the systems’ stability.
CO:SAMPLE_TYPE                   	Tumor cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Pharmaceutical grade asparagine (#1043513, Sigma Aldrich, St Louis, MO, USA) was
TR:TREATMENT_SUMMARY             	injected into the peritoneal cavity twice a week (L-asparagine in phosphate
TR:TREATMENT_SUMMARY             	buffered saline, PBS) at 1 mg/kg and 10 mg/kg body weight for 4 weeks.
TR:TREATMENT_COMPOUND            	asparagine
TR:TREATMENT_ROUTE               	Intraperitoneal
TR:TREATMENT_DOSE                	1 mg/kg and 10 mg/kg asparagine body weight
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Tumor tissue was homogenized in UPLC-grade H2O using the Precellys Evolution
SP:SAMPLEPREP_SUMMARY            	cryo-homogenizer (Bertin Corp. USA) and metabolites were extracted using
SP:SAMPLEPREP_SUMMARY            	methanol:acetonitrile (1:1, v/v). For a comprehensive analysis of the
SP:SAMPLEPREP_SUMMARY            	metabolome, untargeted metabolomics was conducted in-house. Briefly, samples
SP:SAMPLEPREP_SUMMARY            	were analyzed in hydrophilic interaction chromatography (HILIC) and RPLC mass
SP:SAMPLEPREP_SUMMARY            	spectrometry (MS) column chemistry.
SP:PROCESSING_STORAGE_CONDITIONS 	-20℃
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	RP-LCMS
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Waters Acquity H-Class
CH:COLUMN_NAME                   	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
CH:SOLVENT_A                     	0.1% formic acid in water
CH:SOLVENT_B                     	0.1% formic acid in acetonitrile
CH:FLOW_GRADIENT                 	Initial: 99% Solvent A: 1% Solvent B 1 min: 99% Solvent A: 1% Solvent B 8 min:
CH:FLOW_GRADIENT                 	1% Solvent A: 99% Solvent B 10 min: 1% Solvent A: 99% Solvent B 10.10 min: 99%
CH:FLOW_GRADIENT                 	Solvent A: 1% Solvent B 12 min: 99% Solvent A: 1% Solvent B
CH:FLOW_RATE                     	0.5mL/min
CH:COLUMN_TEMPERATURE            	30
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:LABORATORY_NAME               	Johnson lab (Yale University)
AN:OPERATOR_NAME                 	Abhishek Jain
AN:DETECTOR_TYPE                 	W2424, W2475, W2489, W2998
AN:SOFTWARE_VERSION              	v4.2
AN:ACQUISITION_DATE              	September 20th 2023
AN:ACQUISITION_PARAMETERS_FILE   	Polarity: Positive; Analyser mode: Resolution; Dynamic Range: Normal; Target
AN:ACQUISITION_PARAMETERS_FILE   	Enhancement: Off; Source: ES; Acquisition time: 0 min start time, 10 min end
AN:ACQUISITION_PARAMETERS_FILE   	time
AN:DATA_FORMAT                   	.raw
#MS
MS:INSTRUMENT_NAME               	Waters Xevo-G2-XS
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	raw MS data (.raw) were converted to mzML files using ProteoWizard MSConvert
MS:MS_COMMENTS                   	(http://proteowizard.sourceforge.net/). The files were then processed in R
MS:MS_COMMENTS                   	(version 4.2.1) using the XCMS package for feature detection, retention time
MS:MS_COMMENTS                   	correction and alignment
MS:CAPILLARY_VOLTAGE             	2.55 kV
MS:COLLISION_ENERGY              	not applicable
MS:COLLISION_GAS                 	not applicable
MS:MASS_ACCURACY                 	<1ppm error
MS:SOURCE_TEMPERATURE            	150 °C
MS:DESOLVATION_GAS_FLOW          	1000 L/h
MS:DESOLVATION_TEMPERATURE       	500 °C
MS:SCANNING                      	MS Full scan
MS:SCANNING_CYCLE                	300 ms/scan
MS:SCANNING_RANGE                	100–1200 Da
MS:MS_RESULTS_FILE               	ST004470_AN007494_Results.txt	UNITS:Peak Area	Has m/z:Yes	Has RT:Yes	RT units:Seconds
#END