#METABOLOMICS WORKBENCH silviaradenkovic_20230814_142459 DATATRACK_ID:4221 STUDY_ID:ST002816 ANALYSIS_ID:AN004583 PROJECT_ID:PR001759
VERSION             	1
CREATED_ON             	August 17, 2023, 1:19 pm
#PROJECT
PR:PROJECT_TITLE                 	Metabolomic profiling of PMM2-CDG brain organoids
PR:PROJECT_SUMMARY               	PMM2-CDG is a rare inborn error of metabolism caused by deficiency of the PMM
PR:PROJECT_SUMMARY               	enzyme, which leads to impaired protein glycosylation. While the disorder has
PR:PROJECT_SUMMARY               	primarily neurological presentation, there is limited knowledge about the
PR:PROJECT_SUMMARY               	specific brain-related changes that result from PMM deficiency. Utilizing 3D
PR:PROJECT_SUMMARY               	brain organoids derived from individuals with PMM2-CDG, we identified abnormal
PR:PROJECT_SUMMARY               	glucose metabolism in PMM2-CDG organoids, indicating disturbances in metabolic
PR:PROJECT_SUMMARY               	utilization.
PR:INSTITUTE                     	Mayo Clinic
PR:LAST_NAME                     	Radenkovic
PR:FIRST_NAME                    	Silvia
PR:ADDRESS                       	200 2nd Ave SW Rochester MN
PR:EMAIL                         	radenkovic.silvia@mayo.edu
PR:PHONE                         	507(77) 6-6107
PR:FUNDING_SOURCE                	NIH, KU Leuven
#STUDY
ST:STUDY_TITLE                   	Metabolomic profiling of PMM2-CDG brain organoids - Part 2
ST:STUDY_SUMMARY                 	PMM2-CDG is a rare inborn error of metabolism caused by deficiency of the PMM
ST:STUDY_SUMMARY                 	enzyme, which leads to impaired protein glycosylation. While the disorder has
ST:STUDY_SUMMARY                 	primarily neurological presentation, there is limited knowledge about the
ST:STUDY_SUMMARY                 	specific brain-related changes that result from PMM deficiency. Utilizing 3D
ST:STUDY_SUMMARY                 	brain organoids derived from individuals with PMM2-CDG, we identified abnormal
ST:STUDY_SUMMARY                 	glucose metabolism in PMM2-CDG organoids, indicating disturbances in metabolic
ST:STUDY_SUMMARY                 	utilization. In this experiment, day 40 organoids were used.
ST:INSTITUTE                     	Mayo Clinic
ST:LAST_NAME                     	Radenkovic
ST:FIRST_NAME                    	Silvia
ST:ADDRESS                       	200 2nd Ave SW Rochester MN, USA
ST:EMAIL                         	radenkovic.silvia@mayo.edu
ST:PHONE                         	507(77) 6-6107
#SUBJECT
SU:SUBJECT_TYPE                  	Human
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:GENOTYPE_STRAIN               	WT/PMM2-CDG
SU:AGE_OR_AGE_RANGE              	5-7
SU:GENDER                        	Male and female
#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           	8858	S (1)	Genotype:CTR	Cell type=Brain organoids; RAW_FILE_NAME=S (1); DAYS IN VITRO=40; Weight in mg=12
SUBJECT_SAMPLE_FACTORS           	8858	S (2)	Genotype:CTR	Cell type=Brain organoids; RAW_FILE_NAME=S (2); DAYS IN VITRO=40; Weight in mg=12
SUBJECT_SAMPLE_FACTORS           	8858	S (3)	Genotype:CTR	Cell type=Brain organoids; RAW_FILE_NAME=S (3); DAYS IN VITRO=40; Weight in mg=12
SUBJECT_SAMPLE_FACTORS           	8858	S (4)	Genotype:CTR	Cell type=Brain organoids; RAW_FILE_NAME=S (4); DAYS IN VITRO=40; Weight in mg=9.9
SUBJECT_SAMPLE_FACTORS           	8858	S (5)	Genotype:CTR	Cell type=Brain organoids; RAW_FILE_NAME=S (5); DAYS IN VITRO=40; Weight in mg=6.5
SUBJECT_SAMPLE_FACTORS           	1363	S (6)	Genotype:CTR	Cell type=Brain organoids; RAW_FILE_NAME=S (6); DAYS IN VITRO=40; Weight in mg=22
SUBJECT_SAMPLE_FACTORS           	1363	S (7)	Genotype:CTR	Cell type=Brain organoids; RAW_FILE_NAME=S (7); DAYS IN VITRO=40; Weight in mg=22
SUBJECT_SAMPLE_FACTORS           	1363	S (8)	Genotype:CTR	Cell type=Brain organoids; RAW_FILE_NAME=S (8); DAYS IN VITRO=40; Weight in mg=22
SUBJECT_SAMPLE_FACTORS           	1363	S (9)	Genotype:CTR	Cell type=Brain organoids; RAW_FILE_NAME=S (9); DAYS IN VITRO=40; Weight in mg=10.1
SUBJECT_SAMPLE_FACTORS           	1363	S (10)	Genotype:CTR	Cell type=Brain organoids; RAW_FILE_NAME=S (10); DAYS IN VITRO=40; Weight in mg=13.7
SUBJECT_SAMPLE_FACTORS           	P1	S (11)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S (11); DAYS IN VITRO=40; Weight in mg=15
SUBJECT_SAMPLE_FACTORS           	P1	S (12)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S (12); DAYS IN VITRO=40; Weight in mg=15
SUBJECT_SAMPLE_FACTORS           	P1	S (13)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S (13); DAYS IN VITRO=40; Weight in mg=15
SUBJECT_SAMPLE_FACTORS           	P1	S (14)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S (14); DAYS IN VITRO=40; Weight in mg=14.5
SUBJECT_SAMPLE_FACTORS           	P1	S (15)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S (15); DAYS IN VITRO=40; Weight in mg=15.7
SUBJECT_SAMPLE_FACTORS           	P2	S (16)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S (16); DAYS IN VITRO=40; Weight in mg=28
SUBJECT_SAMPLE_FACTORS           	P2	S (17)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S (17); DAYS IN VITRO=40; Weight in mg=28
SUBJECT_SAMPLE_FACTORS           	P2	S (18)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S (18); DAYS IN VITRO=40; Weight in mg=28
SUBJECT_SAMPLE_FACTORS           	P3	S (19)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S (19); DAYS IN VITRO=40; Weight in mg=29.4
SUBJECT_SAMPLE_FACTORS           	P3	S (20)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S (20); DAYS IN VITRO=40; Weight in mg=29.4
SUBJECT_SAMPLE_FACTORS           	P3	S (21)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S (21); DAYS IN VITRO=40; Weight in mg=29.4
#COLLECTION
CO:COLLECTION_SUMMARY            	Brain were collected at day in vitro 160. Briefly, 5 organoids per cell line
CO:COLLECTION_SUMMARY            	were collected and washed three times in DPBS (Gibco). The DPBS was then
CO:COLLECTION_SUMMARY            	removed, the organoids flash frozen and kept in -80 °C prior to metabolomics
CO:COLLECTION_SUMMARY            	experiments. The metabolites were extracted using two phase extraction protocol.
CO:COLLECTION_SUMMARY            	First, the organoids were transferred to lysing matrix tube and 350 µL of
CO:COLLECTION_SUMMARY            	ice-cold extraction buffer (80 % MeOH, IS) was added to the sample. Next, the
CO:COLLECTION_SUMMARY            	organoids were lyzed with ribolyzer, the lysate transferred to 1.5 mL Eppendorf
CO:COLLECTION_SUMMARY            	tube and placed overnight at -80 °C. Further, the samples were centrifuged at
CO:COLLECTION_SUMMARY            	15,000 rpm, 4 °C, 20 min. 100 µL of supernatant was transferred to a fresh
CO:COLLECTION_SUMMARY            	Eppendorf tube and 35 µL of ddH20 was added, followed by 800 µL 100%
CO:COLLECTION_SUMMARY            	chloroform. The samples were then vortexed and stored at 4 °C overnight. Next,
CO:COLLECTION_SUMMARY            	the polar phase was the used for Liquid Chromatography/Mass Spectrometry (LC/MS)
CO:SAMPLE_TYPE                   	Brain organoids
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	NA
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Briefly, 5 organoids per cell line were collected and washed three times in
SP:SAMPLEPREP_SUMMARY            	DPBS. The DPBS was then removed, the organoids flash frozen and kept in -80 °C
SP:SAMPLEPREP_SUMMARY            	prior to metabolomics experiments. The metabolites were extracted using two
SP:SAMPLEPREP_SUMMARY            	phase extraction protocol. First, the organoids were transferred to lysing
SP:SAMPLEPREP_SUMMARY            	matrix tube and 350 µL of ice-cold extraction buffer (80 % MeOH, IS) was added
SP:SAMPLEPREP_SUMMARY            	to the sample. Next, the organoids were lyzed with ribolyzer, the lysate
SP:SAMPLEPREP_SUMMARY            	transferred to 1.5 mL Eppendorf tube and placed overnight at -80 °C. Further,
SP:SAMPLEPREP_SUMMARY            	the samples were centrifuged at 15,000 rpm, 4 °C, 20 min. 100 µL of
SP:SAMPLEPREP_SUMMARY            	supernatant was transferred to a fresh Eppendorf tube and 35 µL of ddH20 was
SP:SAMPLEPREP_SUMMARY            	added, followed by 800 µL 100% chloroform. The samples were then vortexed and
SP:SAMPLEPREP_SUMMARY            	stored at 4 °C overnight. Next, the polar phase was the used for Liquid
SP:SAMPLEPREP_SUMMARY            	Chromatography/Mass Spectrometry (LC/MS)
SP:PROCESSING_STORAGE_CONDITIONS 	-80℃
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	C18 iP REVERSE PHASE
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Waters Acquity
CH:COLUMN_NAME                   	Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um)
CH:SOLVENT_A                     	100% water; 10mM tributylamine; 15mM acetic acid
CH:SOLVENT_B                     	100% methanol
CH:FLOW_GRADIENT                 	The gradient started with 5% of solvent B and 95% solvent A and remained at 5% B
CH:FLOW_GRADIENT                 	until 2 min post injection. A linear gradient to 37% B was carried out until 7
CH:FLOW_GRADIENT                 	min and increased to 41% until 14 min. Between 14 and 26 minutes the gradient
CH:FLOW_GRADIENT                 	increased to 95% of B and remained at 95% B for 4 minutes. At 30 min the
CH:FLOW_GRADIENT                 	gradient returned to 5% B. The chromatography was stopped at 40 min
CH:FLOW_RATE                     	0.25 ml/min
CH:COLUMN_TEMPERATURE            	40
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	El-Maven polly, ThermoFisher Xcalibur, Metabolites were annotated based on the
MS:MS_COMMENTS                   	in-house metabolite library- elution time and m/z values
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	AUC
MS_METABOLITE_DATA_START
Samples	S (1)	S (2)	S (3)	S (4)	S (5)	S (6)	S (7)	S (8)	S (9)	S (10)	S (11)	S (12)	S (13)	S (14)	S (15)	S (16)	S (17)	S (18)	S (19)	S (20)	S (21)
Factors	Genotype:CTR	Genotype:CTR	Genotype:CTR	Genotype:CTR	Genotype:CTR	Genotype:CTR	Genotype:CTR	Genotype:CTR	Genotype:CTR	Genotype:CTR	Genotype:PMM2-CDG	Genotype:PMM2-CDG	Genotype:PMM2-CDG	Genotype:PMM2-CDG	Genotype:PMM2-CDG	Genotype:PMM2-CDG	Genotype:PMM2-CDG	Genotype:PMM2-CDG	Genotype:PMM2-CDG	Genotype:PMM2-CDG	Genotype:PMM2-CDG
13C5-D5-15N Glutamic acid	173146528	180944384	181920528	9.77E+07	5.95E+07	173586048	169469952	173499040	7.04E+07	8.66E+07	153715424	154236992	153976352	1.16E+08	1.16E+08	157056400	156501760	153445328	158393792	156283600	162014768
GDP-mannose	1.73E+07	1.74E+07	1.73E+07	1.77E+06	8.58E+05	4.76E+07	4.95E+07	4.87E+07	7.34E+05	1.61E+06	5.63E+06	5.39E+06	5.97E+06	1.52E+06	1.96E+06	2.87E+07	2.73E+07	2.74E+07	4.53E+07	4.43E+07	4.19E+07
Glucose	3.04E+08	3.32E+08	3.16E+08	3.37E+08	2.96E+08	4.43E+08	4.47E+08	4.51E+08	4.80E+08	5.14E+08	1.63E+08	1.61E+08	1.64E+08	2.10E+08	1.73E+08	3.31E+08	3.23E+08	3.23E+08	3.06E+08	3.15E+08	3.19E+08
Glucose-6-P	7.19E+07	7.68E+07	7.58E+07	2.90E+06	9.00E+06	3.06E+08	3.17E+08	3.24E+08	9.20E+05	5.61E+06	3.70E+07	3.77E+07	3.83E+07	3.10E+07	9.30E+07	4.69E+08	4.79E+08	4.84E+08	5.34E+08	5.49E+08	5.44E+08
Lactate	9.44E+09	9.78E+09	9.83E+09	3.63E+09	2.16E+09	1.44E+10	1.47E+10	1.44E+10	3.42E+09	3.75E+09	1.11E+10	1.13E+10	1.14E+10	4.08E+09	5.24E+09	1.58E+10	1.59E+10	1.60E+10	1.39E+10	1.43E+10	1.41E+10
Sorbitol	4.37E+07	4.38E+07	4.47E+07	9.83E+06	9.29E+06	4.14E+07	4.31E+07	4.46E+07	1.26E+07	1.28E+07	2.28E+07	2.33E+07	2.34E+07	1.20E+07	1.05E+07	2.94E+07	3.12E+07	3.01E+07	2.65E+07	2.71E+07	2.84E+07
Pyruvate	4.71E+08	4.74E+08	4.73E+08	1.55E+07	7.02E+06	1.60E+09	1.56E+09	1.54E+09	3.68E+07	3.19E+07	6.01E+08	5.96E+08	5.93E+08	3.28E+07	4.53E+07	1.35E+09	1.33E+09	1.33E+09	1.20E+09	1.19E+09	1.17E+09
Taurine	4.99E+08	5.12E+08	5.21E+08	1.00E+08	9.51E+07	6.97E+08	7.70E+08	7.63E+08	1.10E+08	1.03E+08	2.02E+08	2.11E+08	2.21E+08	2.40E+08	2.72E+08	6.17E+08	6.43E+08	6.42E+08	6.23E+08	6.17E+08	6.15E+08
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	parent formula	M/Z	HMDB ID
13C5-D5-15N Glutamic acid	HOO13C13CD213CD213CD(15NH2)13COOH	157.09107	329766021
GDP-mannose	C16H25N5O16P2	604.06988	HMDB01163
Glucose	C6H12O6	179.05611	HMDB00122
Glucose-6-P	C6H13O9P	259.02244	HMDB01401
Lactate	C3H6O3	89.02442	HMDB00190
Sorbitol	C6H14O6	181.07164	HMDB00247
Pyruvate	C3H4O3	87.00877	HMDB00243
Taurine	C2H7NO3S	124.00739	HMDB00251
METABOLITES_END
#END