#METABOLOMICS WORKBENCH silviaradenkovic_20230814_141643 DATATRACK_ID:4220 STUDY_ID:ST002812 ANALYSIS_ID:AN004579 PROJECT_ID:PR001759
VERSION             	1
CREATED_ON             	August 15, 2023, 5:13 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 1
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 160 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           	1363	S01 (1)	Genotype:CTR	Cell type=Brain organoids; RAW_FILE_NAME=S01 (1); DAYS IN VITRO=160; Weight in mg=4.4
SUBJECT_SAMPLE_FACTORS           	1363	S01 (2)	Genotype:CTR	Cell type=Brain organoids; RAW_FILE_NAME=S01 (2); DAYS IN VITRO=160; Weight in mg=4.5
SUBJECT_SAMPLE_FACTORS           	8858	S01 (3)	Genotype:CTR	Cell type=Brain organoids; RAW_FILE_NAME=S01 (3); DAYS IN VITRO=160; Weight in mg=3.4
SUBJECT_SAMPLE_FACTORS           	8858	S01 (4)	Genotype:CTR	Cell type=Brain organoids; RAW_FILE_NAME=S01 (4); DAYS IN VITRO=160; Weight in mg=4.8
SUBJECT_SAMPLE_FACTORS           	8858	S01 (5)	Genotype:CTR	Cell type=Brain organoids; RAW_FILE_NAME=S01 (5); DAYS IN VITRO=160; Weight in mg=4.1
SUBJECT_SAMPLE_FACTORS           	P2	S01 (6)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S01 (6); DAYS IN VITRO=160; Weight in mg=5.6
SUBJECT_SAMPLE_FACTORS           	P2	S01 (7)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S01 (7); DAYS IN VITRO=160; Weight in mg=5.3
SUBJECT_SAMPLE_FACTORS           	P2	S01 (8)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S01 (8); DAYS IN VITRO=160; Weight in mg=11.4
SUBJECT_SAMPLE_FACTORS           	P2	S01 (9)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S01 (9); DAYS IN VITRO=160; Weight in mg=9.6
SUBJECT_SAMPLE_FACTORS           	P1	S01 (10)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S01 (10); DAYS IN VITRO=160; Weight in mg=6.1
SUBJECT_SAMPLE_FACTORS           	P1	S01 (11)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S01 (11); DAYS IN VITRO=160; Weight in mg=9.5
SUBJECT_SAMPLE_FACTORS           	P1	S01 (12)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S01 (12); DAYS IN VITRO=160; Weight in mg=7.5
SUBJECT_SAMPLE_FACTORS           	P1	S01 (13)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S01 (13); DAYS IN VITRO=160; Weight in mg=13.2
SUBJECT_SAMPLE_FACTORS           	P3	S01 (14)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S01 (14); DAYS IN VITRO=160; Weight in mg=6.6
SUBJECT_SAMPLE_FACTORS           	P3	S01 (15)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S01 (15); DAYS IN VITRO=160; Weight in mg=4.4
SUBJECT_SAMPLE_FACTORS           	P3	S01 (16)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S01 (16); DAYS IN VITRO=160; Weight in mg=6.3
SUBJECT_SAMPLE_FACTORS           	P3	S01 (17)	Genotype:PMM2-CDG	Cell type=Brain organoids; RAW_FILE_NAME=S01 (17); DAYS IN VITRO=160; Weight in mg=7.5
#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	S01 (1)	S01 (2)	S01 (3)	S01 (4)	S01 (5)	S01 (6)	S01 (7)	S01 (8)	S01 (9)	S01 (10)	S01 (11)	S01 (12)	S01 (13)	S01 (14)	S01 (15)	S01 (16)	S01 (17)
Factors	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	Genotype:PMM2-CDG
13C5-D5-15N Glutamic acid	3.20E+08	2.36E+08	4.24E+08	3.10E+08	1.32E+08	3.82E+08	7.91E+07	4.46E+08	4.51E+08	4.79E+08	4.81E+08	4.76E+08	4.69E+08	4.62E+08	4.06E+08	4.48E+08	4.53E+08
GDP-mannose	1.18E+06	6.80E+05	9.26E+05	4.36E+05	3.03E+04	4.20E+06	3.52E+05	4.06E+06	1.16E+06	4.42E+06	2.35E+06	2.77E+06	5.54E+06	2.07E+06	5.29E+06	3.72E+06	7.37E+06
Glucose	1.73E+08	1.88E+08	7.25E+07	1.27E+08	9.22E+07	1.50E+08	5.35E+07	1.06E+08	8.39E+07	3.68E+08	3.63E+08	5.19E+08	2.91E+08	1.73E+08	2.96E+08	2.13E+08	2.95E+08
Glucose-6-P	8.22E+06	5.43E+06	1.56E+07	9.63E+06	5.35E+06	4.87E+08	3.48E+06	5.53E+07	1.70E+08	1.30E+09	4.61E+08	5.94E+08	1.10E+09	2.12E+08	2.30E+08	1.79E+07	5.70E+08
Lactate	2.69E+09	3.14E+09	2.77E+09	2.26E+09	1.79E+09	3.98E+09	2.16E+09	5.42E+09	2.91E+09	3.84E+09	3.02E+09	3.36E+09	3.64E+09	3.15E+09	3.52E+09	4.77E+09	1.09E+10
Sorbitol	8.41E+07	1.00E+08	7.80E+07	1.81E+08	7.80E+07	1.12E+08	2.90E+07	1.08E+08	1.26E+08	6.48E+07	7.70E+07	6.53E+07	1.26E+08	1.05E+08	8.44E+07	7.72E+07	5.13E+07
Pyruvate	7.32E+08	7.59E+08	6.74E+08	6.64E+08	6.74E+08	7.51E+08	8.47E+08	7.77E+08	7.11E+08	8.86E+08	9.22E+08	9.68E+08	9.39E+08	8.95E+08	8.76E+08	9.41E+08	1.02E+09
Taurine	6.23E+08	4.49E+08	3.77E+08	4.15E+08	2.85E+08	2.11E+08	8.53E+07	1.93E+08	1.34E+08	8.39E+08	8.65E+08	8.99E+08	7.26E+08	5.11E+08	5.51E+08	6.58E+08	6.33E+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