#METABOLOMICS WORKBENCH estancliffe_20230324_124549 DATATRACK_ID:3819 STUDY_ID:ST002534 ANALYSIS_ID:AN004169 PROJECT_ID:PR001630
VERSION             	1
CREATED_ON             	March 29, 2023, 12:31 pm
#PROJECT
PR:PROJECT_TITLE                 	Using Mass Spectrometry Imaging to Map Fluxes Quantitatively in the Tumor
PR:PROJECT_TITLE                 	Ecosystem
PR:PROJECT_SUMMARY               	Tumors are comprised of a multitude of cell types spanning different
PR:PROJECT_SUMMARY               	microenvironments. Mass spectrometry imaging (MSI) has the potential to identify
PR:PROJECT_SUMMARY               	metabolic patterns within the tumor ecosystem and surrounding tissues, but
PR:PROJECT_SUMMARY               	conventional workflows have not yet fully integrated the breadth of experimental
PR:PROJECT_SUMMARY               	techniques in metabolomics. Here, we combine MSI, stable isotope labeling, and a
PR:PROJECT_SUMMARY               	spatial variant of Isotopologue Spectral Analysis to map distributions of
PR:PROJECT_SUMMARY               	metabolite abundances, nutrient contributions, and metabolic turnover fluxes
PR:PROJECT_SUMMARY               	across the brains of mice harboring GL261 glioma, a widely used model for
PR:PROJECT_SUMMARY               	glioblastoma. When integrated with MSI, the combination of ion mobility,
PR:PROJECT_SUMMARY               	Desorption Electrospray Ionization, and Matrix Assisted Laser Desorption
PR:PROJECT_SUMMARY               	revealed disruption in multiple anabolic pathways. De novo fatty acid synthesis
PR:PROJECT_SUMMARY               	flux was determined to be increased by approximately 3-fold in glioma relative
PR:PROJECT_SUMMARY               	to surrounding healthy tissue. Fatty acid elongation flux was elevated even
PR:PROJECT_SUMMARY               	higher at 8-fold and highlights the importance of elongase activity in glioma.
PR:PROJECT_SUMMARY               	The fluxes we examined were uniformly increased throughout the entire tumor
PR:PROJECT_SUMMARY               	region, revealing a high degree of metabolic homogeneity in our model of
PR:PROJECT_SUMMARY               	glioblastoma.
PR:INSTITUTE                     	Washington University in St. Louis
PR:LAST_NAME                     	Stancliffe
PR:FIRST_NAME                    	Ethan
PR:ADDRESS                       	1 Brookings Dr. Campus Box 1134, St. Louis, MO 63105
PR:EMAIL                         	estancliffe@wustl.edu
PR:PHONE                         	3194644881
#STUDY
ST:STUDY_TITLE                   	Using Mass Spectrometry Imaging to Map Fluxes Quantitatively in the Tumor
ST:STUDY_TITLE                   	Ecosystem
ST:STUDY_SUMMARY                 	Tumors are comprised of a multitude of cell types spanning different
ST:STUDY_SUMMARY                 	microenvironments. Mass spectrometry imaging (MSI) has the potential to identify
ST:STUDY_SUMMARY                 	metabolic patterns within the tumor ecosystem and surrounding tissues, but
ST:STUDY_SUMMARY                 	conventional workflows have not yet fully integrated the breadth of experimental
ST:STUDY_SUMMARY                 	techniques in metabolomics. Here, we combine MSI, stable isotope labeling, and a
ST:STUDY_SUMMARY                 	spatial variant of Isotopologue Spectral Analysis to map distributions of
ST:STUDY_SUMMARY                 	metabolite abundances, nutrient contributions, and metabolic turnover fluxes
ST:STUDY_SUMMARY                 	across the brains of mice harboring GL261 glioma, a widely used model for
ST:STUDY_SUMMARY                 	glioblastoma. When integrated with MSI, the combination of ion mobility,
ST:STUDY_SUMMARY                 	Desorption Electrospray Ionization, and Matrix Assisted Laser Desorption
ST:STUDY_SUMMARY                 	revealed disruption in multiple anabolic pathways. De novo fatty acid synthesis
ST:STUDY_SUMMARY                 	flux was determined to be increased by approximately 3-fold in glioma relative
ST:STUDY_SUMMARY                 	to surrounding healthy tissue. Fatty acid elongation flux was elevated even
ST:STUDY_SUMMARY                 	higher at 8-fold and highlights the importance of elongase activity in glioma.
ST:STUDY_SUMMARY                 	The fluxes we examined were uniformly increased throughout the entire tumor
ST:STUDY_SUMMARY                 	region, revealing a high degree of metabolic homogeneity in our model of
ST:STUDY_SUMMARY                 	glioblastoma.
ST:INSTITUTE                     	Washington University in St. Louis
ST:DEPARTMENT                    	Chemistry
ST:LABORATORY                    	Patti
ST:LAST_NAME                     	Stancliffe
ST:FIRST_NAME                    	Ethan
ST:ADDRESS                       	1 Brookings Dr. Campus Box 1134, St. Louis, MO 63105
ST:EMAIL                         	estancliffe@wustl.edu
ST:PHONE                         	3194644881
ST:NUM_GROUPS                    	2
ST:TOTAL_SUBJECTS                	8
ST:NUM_FEMALES                   	8
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
#FACTORS
#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           	-	4-1_serum	Sample type:serum | Isotopically labeled:yes | Group:serum	RAW_FILE_NAME=4-1_serum_pns.mzML
SUBJECT_SAMPLE_FACTORS           	-	4-2_serum	Sample type:serum | Isotopically labeled:yes | Group:serum	RAW_FILE_NAME=4-2_serum_pns.mzML
SUBJECT_SAMPLE_FACTORS           	-	4-3_serum	Sample type:serum | Isotopically labeled:yes | Group:serum	RAW_FILE_NAME=4-3_serum_pns.mzML
SUBJECT_SAMPLE_FACTORS           	-	4-4_serum	Sample type:serum | Isotopically labeled:yes | Group:serum	RAW_FILE_NAME=4-4_serum_pns.mzML
SUBJECT_SAMPLE_FACTORS           	-	1-1_tumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor	RAW_FILE_NAME=pHILIC_Brain1-1_LH5.mzML
SUBJECT_SAMPLE_FACTORS           	-	1-1_nontumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor	RAW_FILE_NAME=pHILIC_Brain1-1_RH5.mzML
SUBJECT_SAMPLE_FACTORS           	-	1-3_tumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor	RAW_FILE_NAME=pHILIC_Brain1-3_LH5.mzML
SUBJECT_SAMPLE_FACTORS           	-	1-3_nontumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor	RAW_FILE_NAME=pHILIC_Brain1-3_RH5.mzML
SUBJECT_SAMPLE_FACTORS           	-	1-5_tumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor	RAW_FILE_NAME=pHILIC_Brain1-5_LH5.mzML
SUBJECT_SAMPLE_FACTORS           	-	1-5_nontumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor	RAW_FILE_NAME=pHILIC_Brain1-5_RH5.mzML
SUBJECT_SAMPLE_FACTORS           	-	2-3_tumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor	RAW_FILE_NAME=pHILIC_Brain2-3_LH5.mzML
SUBJECT_SAMPLE_FACTORS           	-	2-3_nontumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor	RAW_FILE_NAME=pHILIC_Brain2-3_RH5.mzML
SUBJECT_SAMPLE_FACTORS           	-	2-5_tumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor	RAW_FILE_NAME=pHILIC_Brain2-5_LH5.mzML
SUBJECT_SAMPLE_FACTORS           	-	2-5_nontumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor	RAW_FILE_NAME=pHILIC_Brain2-5_RH5.mzML
SUBJECT_SAMPLE_FACTORS           	-	4-1_tumor	Sample type:brain tissue | Isotopically labeled:yes | Group:brain_tumor	RAW_FILE_NAME=pHILIC_Brain4-1_LH2.mzML
SUBJECT_SAMPLE_FACTORS           	-	4-1_nontumor	Sample type:brain tissue | Isotopically labeled:yes | Group:brain_nontumor	RAW_FILE_NAME=pHILIC_Brain4-1_RH2.mzML
SUBJECT_SAMPLE_FACTORS           	-	4-2_tumor	Sample type:brain tissue | Isotopically labeled:yes | Group:brain_tumor	RAW_FILE_NAME=pHILIC_Brain4-2_LH1.mzML
SUBJECT_SAMPLE_FACTORS           	-	4-2_nontumor	Sample type:brain tissue | Isotopically labeled:yes | Group:brain_nontumor	RAW_FILE_NAME=pHILIC_Brain4-2_RH1.mzML
SUBJECT_SAMPLE_FACTORS           	-	4-3_tumor	Sample type:brain tissue | Isotopically labeled:yes | Group:brain_tumor	RAW_FILE_NAME=pHILIC_Brain4-3_LH2.mzML
SUBJECT_SAMPLE_FACTORS           	-	4-3_nontumor	Sample type:brain tissue | Isotopically labeled:yes | Group:brain_nontumor	RAW_FILE_NAME=pHILIC_Brain4-3_RH2.mzML
SUBJECT_SAMPLE_FACTORS           	-	4-4_tumor	Sample type:brain tissue | Isotopically labeled:yes | Group:brain_tumor	RAW_FILE_NAME=pHILIC_Brain4-4_LH4.mzML
SUBJECT_SAMPLE_FACTORS           	-	4-4_nontumor	Sample type:brain tissue | Isotopically labeled:yes | Group:brain_nontumor	RAW_FILE_NAME=pHILIC_Brain4-4_RH4.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	Brains were embedded in 5% wt. carboxymethyl cellulose (Millipore Sigma) in
CO:COLLECTION_SUMMARY            	water and stored at -80 °C. Next, 20 µm thick sections were collected at -20
CO:COLLECTION_SUMMARY            	°C by using a CM1860 cryostat (Leica Biosystems). Superfrost Plus slides
CO:COLLECTION_SUMMARY            	(Thermo Fisher Scientific) were used after cleaning with ethanol. Sections were
CO:COLLECTION_SUMMARY            	dried under vacuum, stored at -80 °C until use, and thawed under vacuum
CO:COLLECTION_SUMMARY            	immediately prior to analysis. For MALDI, 10 µm thick sections were collected
CO:COLLECTION_SUMMARY            	on SiO2 passivated, indium tin oxide coated polished float glass slides (Delta
CO:COLLECTION_SUMMARY            	Technologies Limited, Loveland, CO, USA). Serial 50 µm thick sections were
CO:COLLECTION_SUMMARY            	collected for extraction and LC/MS analysis after dividing between
CO:COLLECTION_SUMMARY            	tumor-containing and non-tumor hemispheres (as described below).
CO:SAMPLE_TYPE                   	Brain
#TREATMENT
TR:TREATMENT_SUMMARY             	No treatments were applied.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Sections of 50 µm brain tissue were cut into left (tumor) and right (non-tumor)
SP:SAMPLEPREP_SUMMARY            	hemispheres with a razor blade and collected into Eppendorf tubes while being
SP:SAMPLEPREP_SUMMARY            	kept frozen in the cryostat. The samples were then extracted with 2:2:1
SP:SAMPLEPREP_SUMMARY            	acetonitrile, methanol, water at a ratio of 80 µL per mg wet weight. The weight
SP:SAMPLEPREP_SUMMARY            	was calculated based on area and thickness of the sections. The solvent was
SP:SAMPLEPREP_SUMMARY            	added to the tissue and vigorously vortexed. For serum analysis, 5 µL serum was
SP:SAMPLEPREP_SUMMARY            	mixed with 45 µL of a 4:4:1 mixture of acetonitrile, methanol, water and
SP:SAMPLEPREP_SUMMARY            	vortexed. Extraction blanks were prepared with 5 µL water instead of serum. All
SP:SAMPLEPREP_SUMMARY            	samples were kept at -20 °C overnight. After centrifugation at 14,000 g for 10
SP:SAMPLEPREP_SUMMARY            	min at 4 °C, the supernatant was transferred to an LC/MS vial.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Metabolites were separated via hydrophilic interaction liquid chromatography
CH:CHROMATOGRAPHY_SUMMARY        	(HILIC) by using a SeQuant ZIC-pHILIC column (100 x 2.1 mm, 5 µm, polymer,
CH:CHROMATOGRAPHY_SUMMARY        	Merck-Millipore) with a ZIC-pHILIC guard column (20 mm x 2.1 mm, 5 µm). The
CH:CHROMATOGRAPHY_SUMMARY        	column compartment temperature was 40 °C and the flow rate was set to 250 µL
CH:CHROMATOGRAPHY_SUMMARY        	min-1. The mobile phases consisted of A: 95% water, 5% acetonitrile, 20 mM
CH:CHROMATOGRAPHY_SUMMARY        	ammonium bicarbonate, 0.1% ammonium hydroxide solution (25% ammonia in water), 5
CH:CHROMATOGRAPHY_SUMMARY        	µM medronic acid; and B: 95% acetonitrile, 5% water. The following linear
CH:CHROMATOGRAPHY_SUMMARY        	gradient was applied: 0 to 1 min, 90% B; 12 min, 35% B; 12.5 to 14.5 min, 25% B;
CH:CHROMATOGRAPHY_SUMMARY        	15 min, 90% B followed by a re-equilibration phase of 4 min at 400 µL min-1 and
CH:CHROMATOGRAPHY_SUMMARY        	2 min at 250 µL min-1. The samples were kept at 6 °C in the autosampler. The
CH:CHROMATOGRAPHY_SUMMARY        	injection volume was 5 µL.
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	SeQuant ZIC-HILIC (150 x 2.1mm,5um)
CH:SOLVENT_A                     	95% water, 5% acetonitrile, 20 mM ammonium bicarbonate, 0.1% ammonium hydroxide
CH:SOLVENT_A                     	solution (25% ammonia in water), 5 µM medronic acid
CH:SOLVENT_B                     	95% acetonitrile, 5% water
CH:FLOW_GRADIENT                 	0 to 1 min, 90% B; 12 min, 35% B; 12.5 to 14.5 min, 25% B; 15 min, 90% B
CH:FLOW_GRADIENT                 	followed by a re-equilibration phase of 4 min at 400 µL min-1 and 2 min at 250
CH:FLOW_GRADIENT                 	µL min-1
CH:FLOW_RATE                     	250 µL min-1
CH:COLUMN_TEMPERATURE            	40 °C
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Orbitrap ID-X tribrid
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	UNSPECIFIED
MS:MS_COMMENTS                   	A Vanquish UHPLC system was coupled to an Orbitrap ID-X Tribrid mass
MS:MS_COMMENTS                   	spectrometer (Thermo Fisher Scientific) via electrospray ionization with the
MS:MS_COMMENTS                   	following source conditions: sheath gas flow 50 arbitrary units (Arb), auxiliary
MS:MS_COMMENTS                   	gas flow 10 Arb, sweep gas flow 1 Arb, ion transfer tube temperature 300 °C,
MS:MS_COMMENTS                   	vaporizer temperature 200 °C respectively. The RF lens value was 60%. Data were
MS:MS_COMMENTS                   	acquired in negative and positive polarity with a spray voltage of 2.8 kV and
MS:MS_COMMENTS                   	3.5 kV, respectively. MS1 data were acquired from 67-900 m/z at a resolution of
MS:MS_COMMENTS                   	120,000 with an automatic gain control (AGC) target of 2e5 and a maximum
MS:MS_COMMENTS                   	injection time of 200 ms in polarity switching mode. MS2 data for metabolite
MS:MS_COMMENTS                   	identification were acquired at a resolution of 15,000 with an AGC target of
MS:MS_COMMENTS                   	2.5e4 and a maximum injection time of 70 ms in negative and positive mode
MS:MS_COMMENTS                   	separately. A 5 ppm mass error and 10 s dynamic exclusion were applied.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Peak areas
MS_METABOLITE_DATA_START
Samples	1-1_tumor	1-1_nontumor	1-3_tumor	1-3_nontumor	1-5_tumor	1-5_nontumor	2-3_tumor	2-3_nontumor	2-5_tumor	2-5_nontumor	4-1_tumor	4-1_nontumor	4-2_tumor	4-2_nontumor	4-3_tumor	4-3_nontumor	4-4_tumor	4-4_nontumor	4-1_serum	4-2_serum	4-3_serum	4-4_serum
Factors	Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor	Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor	Sample type:brain tissue | Isotopically labeled:yes | Group:brain_tumor	Sample type:brain tissue | Isotopically labeled:yes | Group:brain_nontumor	Sample type:brain tissue | Isotopically labeled:yes | Group:brain_tumor	Sample type:brain tissue | Isotopically labeled:yes | Group:brain_nontumor	Sample type:brain tissue | Isotopically labeled:yes | Group:brain_tumor	Sample type:brain tissue | Isotopically labeled:yes | Group:brain_nontumor	Sample type:brain tissue | Isotopically labeled:yes | Group:brain_tumor	Sample type:brain tissue | Isotopically labeled:yes | Group:brain_nontumor	Sample type:serum | Isotopically labeled:yes | Group:serum	Sample type:serum | Isotopically labeled:yes | Group:serum	Sample type:serum | Isotopically labeled:yes | Group:serum	Sample type:serum | Isotopically labeled:yes | Group:serum
Aspartate	49890591	56680348	65606901	72920147	41265543	40551848	41293916	47634054	50768538	52252654	82450479	87193499	54112204	110856241	75801717	88420280	61787641	68207425	19546771	18791006	26469805	31244547
Citrate	61614858	39917573	59244619	58022323	66913864	51475556	54675293	43015171	58566533	48840907	57248390	73794138	55286581	84133615	59217951	73234446	58776580	49245436	191645105	293822408	264927111	183926962
N-actylasparate	713745151	865968393	949131996	969317235	387901312	586116361	546622245	677592843	607496898	721316986	1507728368	1566929856	872821112	1387742400	1290416954	1460698880	915698200	1347663584	1568528	1785828	5810129	2267476
Ribose 5-phosphate	5889548	4242044	4292110	4158670	4169047	3231357	3171181	2885488	5429669	3823249	4433524	4633286	4597684	6734628	5255441	6911781	3809251	2748154	918343	379433	7154108	7764144
AMP	116078523	97172267	140770516	122900184	82970541	64317993	74392530	78910510	112758371	108837245	78649251	77427287	69657050	68769341	77934278	96937396	89056441	70235821	21803	9913	13176	14023
Palmitoylcarnitine	129252520	57000622	109944414	82565954	56464902	15983096	43975388	16543022	65160272	32027846	76696240	39120816	168718168	78183848	51214926	33307574	56097685	22358310	28822824	342064279	38083719	22209631
16:0 Palmitic acid	675917839	709497980	569425556	580512216	427159031	495218216	368844684	396012211	515519443	484230053	606916307	549043485	646765543	656912662	503275228	579309850	436937319	434141818	4018102178	8434115568	4199479608	2708062872
20:4 Arachidonic acid	938710833	1247880860	1036237671	1216912072	578554233	886139045	625089095	797721822	737684200	1013508593	1092652642	1215744662	602540159	1037715278	960991392	1309349390	632443194	964167652	715070530	489263085	656292695	538638771
18:0 Stearic acid	668831865	825063254	667338069	737700691	416582900	577562001	385020868	493976483	540141128	626967477	690614001	724541041	483399398	648820390	575850686	775448588	416174192	559151385	1666013118	1641766468	1184250539	1211909887
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Standardized name	Formula	Exact mass
Aspartate	Aspartic acid	C4H7NO4	133.0375
Citrate	Citric acid	C6H8O7	192.027
N-actylasparate	N-Acetylaspartic acid	C6H9NO5	175.0481
Ribose 5-phosphate	Ribose 5-phosphate	C5H11O8P	230.0192
AMP	AMP	C10H14N5O7P	347.0631
Palmitoylcarnitine	CAR 16:0	C23H45NO4	399.3349
16:0 Palmitic acid	Palmitic acid	C16H32O2	256.2402
20:4 Arachidonic acid	Arachidonic acid	C20H32O2	304.2402
18:0 Stearic acid	Stearic acid	C18H36O2	284.2715
METABOLITES_END
#END