Summary of Study ST003499

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002082. The data can be accessed directly via it's Project DOI: 10.21228/M8H24R This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003499
Study Title	Metabolic analysis of ADSL deficiency patients' EBV-LCLs.
Study Summary	Adenylosuccinate lyase (ADSL) deficiency (ADSLd) is a rare autosomal recessive defect of purine metabolism associated with a wide range of clinical manifestations. Despite ADSL implication in purine synthesis, no additional molecular alterations have been identified as a cause of ADSLd besides the accumulation of toxic substrates. Here we uncover a novel association between ADSLd and mitochondrial dysfunction, which is characterized by an increase in fragmentation and reduction in respiration and ATP production. The extent of this mitochondrial dysfunction is directly proportional to the pathological manifestations of ADSLd, which are predominantly observed in tissues that rely heavily on mitochondria. Our analysis also unveils a striking defect in mitochondrial dynamics and transport, which are associated with the suppression of ERK2 and AKT function. Remarkably, the mitochondrial phenotype can be rescued in part by overexpression of a constitutive form of ERK2 or through the administration of purine intermediates. This scenario provides an alternative explanation of ADSLd onset, reorienting research towards developing innovative therapeutic strategies based on the restoration of mitochondrial metabolism.
Institute	Catholic University of the Sacred Heart
Last Name	Bordi
First Name	Matteo
Address	Largo Francesco Vito 1, Rome, Italy, 00168, Italy
Email	matteo.bordi@unicatt.it
Phone	+390630155135/5258
Submit Date	2024-09-20
Study Comments	This new submission is part of PR002082
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2024-10-16
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002082
Project DOI:	doi: 10.21228/M8H24R
Project Title:	ADSL deficiency drives mitochondrial dysfunction and ERK2 dysregulation in a linear genotype to phenotype correlation
Project Summary:	Adenylosuccinate lyase (ADSL) deficiency (ADSLd) is a rare autosomal recessive defect of purine metabolism associated with a wide range of clinical manifestations. Despite ADSL implication in purine synthesis, no additional molecular alterations have been identified as a cause of ADSLd besides the accumulation of toxic substrates. Here we uncover a novel association between ADSLd and mitochondrial dysfunction, which is characterized by an increase in fragmentation and reduction in respiration and ATP production. The extent of this mitochondrial dysfunction is directly proportional to the pathological manifestations of ADSLd, which are predominantly observed in tissues that rely heavily on mitochondria. Our analysis also unveils a striking defect in mitochondrial dynamics and transport, which are associated with the suppression of ERK2 and AKT function. Remarkably, the mitochondrial phenotype can be rescued in part by overexpression of a constitutive form of ERK2 or through the administration of purine intermediates. This scenario provides an alternative explanation of ADSLd onset, reorienting research towards developing innovative therapeutic strategies based on the restoration of mitochondrial metabolism.
Institute:	Catholic University of the Sacred Heart
Last Name:	Bordi
First Name:	Matteo
Address:	Largo Francesco Vito 1, Rome, Italy, 00168, Italy
Email:	matteo.bordi@unicatt.it
Phone:	+390630155135/5258

Subject:

Subject ID:	SU003627
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Subject ID:	SU003628
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Phenotype	Genotype
SA385661	BT03-001	Blood	Control	Wildtype
SA385662	BT03-002	Blood	Control	Wildtype
SA385663	BT03-003	Blood	Control	Wildtype
SA385664	BT03-004	Blood	Control	Wildtype
SA385665	BT03-005	Blood	Control	Wildtype
SA385676	BT03-001	Blood	Control	Wildtype
SA385677	BT03-002	Blood	Control	Wildtype
SA385678	BT03-003	Blood	Control	Wildtype
SA385679	BT03-004	Blood	Control	Wildtype
SA385680	BT03-005	Blood	Control	Wildtype
SA385666	BT03-011	Blood	Mild	R309H/c.1191+5G->C
SA385667	BT03-012	Blood	Mild	R309H/c.1191+5G->C
SA385668	BT03-013	Blood	Mild	R309H/c.1191+5G->C
SA385669	BT03-014	Blood	Mild	R309H/c.1191+5G->C
SA385670	BT03-015	Blood	Mild	R309H/c.1191+5G->C
SA385681	BT03-011	Blood	Mild	R309H/c.1191+5G->C
SA385682	BT03-012	Blood	Mild	R309H/c.1191+5G->C
SA385683	BT03-013	Blood	Mild	R309H/c.1191+5G->C
SA385684	BT03-014	Blood	Mild	R309H/c.1191+5G->C
SA385685	BT03-015	Blood	Mild	R309H/c.1191+5G->C
SA385671	BT03-006	Blood	Mild	T450S/D332H
SA385672	BT03-007	Blood	Mild	T450S/D332H
SA385673	BT03-008	Blood	Mild	T450S/D332H
SA385674	BT03-009	Blood	Mild	T450S/D332H
SA385675	BT03-010	Blood	Mild	T450S/D332H
SA385686	BT03-006	Blood	Mild	T450S/D332H
SA385687	BT03-007	Blood	Mild	T450S/D332H
SA385688	BT03-008	Blood	Mild	T450S/D332H
SA385689	BT03-009	Blood	Mild	T450S/D332H
SA385690	BT03-010	Blood	Mild	T450S/D332H

Showing results 1 to 30 of 30

Showing results 1 to 30 of 30

Collection:

Collection ID:	CO003620
Collection Summary:	1x106 cells were plated onto 35mm plates in RPMI media (5 replicates for each cell type). After two days, before extraction, cells were counted using CASY cell counter (Omni Life Sciences) using a separate counting plate. After that, cells were washed at room temperature with PBS twice and then kept in a cold bath with dry ice and methanol before adding the metabolite extraction solution.
Sample Type:	Blood (serum)

Collection ID:	CO003621
Collection Summary:	1x106 cells were plated onto 35mm plates in RPMI media (5 replicates for each cell type). After two days, before extraction, cells were counted using CASY cell counter (Omni Life Sciences) using a separate counting plate. After that, cells were washed at room temperature with PBS twice and then kept in a cold bath with dry ice and methanol before adding the metabolite extraction solution.
Sample Type:	Blood (serum)

Treatment:

Treatment ID:	TR003636
Treatment Summary:	Cells were cultured in RPMI media supplemented with 10% FBS (Fetal Bovine Serum)

Treatment ID:	TR003637
Treatment Summary:	Cells were cultured in RPMI media supplemented with 10% FBS (Fetal Bovine Serum)

Sample Preparation:

Sampleprep ID:	SP003634
Sampleprep Summary:	Metabolite extraction solution (50% methanol, 30% acetonitrile, 20% ultrapure water, 5 µM final concentration valine-d8) was added to each well after the washes in PBS following the proportion of 1ml of extraction solution per million cells. The extracts were scraped and mixed at 4°C for 15 min. After final centrifugation at max speed for 15 min at 4°C, the supernatants were transferred into LC-MS vials.

Sampleprep ID:	SP003635
Sampleprep Summary:	Metabolite extraction solution (50% methanol, 30% acetonitrile, 20% ultrapure water, 5 µM final concentration valine-d8) was added to each well after the washes in PBS following the proportion of 1ml of extraction solution per million cells. The extracts were scraped and mixed at 4°C for 15 min. After final centrifugation at max speed for 15 min at 4°C, the supernatants were transferred into LC-MS vials.

Combined analysis:

Analysis ID	AN005743	AN005744
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Dionex Ultimate 3000 UHPLC	Dionex Ultimate 3000 UHPLC
Column	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	UNSPECIFIED	UNSPECIFIED
Units	peak area	peak area

Chromatography:

Chromatography ID:	CH004358
Instrument Name:	Dionex Ultimate 3000 UHPLC
Column Name:	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)
Column Temperature:	40
Flow Gradient:	0-2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-23 min: hold at 80% B.
Flow Rate:	0.200 mL/min
Solvent A:	100% water; 20 mM ammonium carbonate; 0.05% ammonium hydroxide
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

Chromatography ID:	CH004359
Instrument Name:	Dionex Ultimate 3000 UHPLC
Column Name:	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)
Column Temperature:	40
Flow Gradient:	0-2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-23 min: hold at 80% B.
Flow Rate:	0.200 mL/min
Solvent A:	100% water; 20 mM ammonium carbonate; 0.05% ammonium hydroxide
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS005466
Analysis ID:	AN005743
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Metabolites were measured with a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap Mass spectrometer (HRMS) coupled to a Dionex Ultimate 3000 UHPLC. The mass spectrometer was operated in full-scan, polarity-switching mode, with the spray voltage set to +4.5 kV/-3.5 kV, the heated capillary held at 320 °C, and the auxiliary gas heater held at 280 °C. The sheath gas flow was set to 55 units, the auxiliary gas flow was set to 15 units, and the sweep gas flow was set to 0 unit. HRMS data acquisition was performed in a range of m/z = 70–900, with the resolution set at 70,000, the AGC target at 1 × 106, and the maximum injection time (Max IT) at 120 ms. Metabolite identities were confirmed using two parameters: (1) precursor ion m/z was matched within 5 ppm of theoretical mass predicted by the chemical formula; (2) the retention time of metabolites was within 5% of the retention time of a purified standard run with the same chromatographic method. Chromatogram review and peak area integration were performed using the Thermo Fisher software Tracefinder 5.0 and the peak area for each detected metabolite was normalized against the total ion count (TIC) of that sample to correct any variations introduced from sample handling through instrument analysis. The normalized areas were used as variables for further statistical data analysis.
Ion Mode:	UNSPECIFIED

MS ID:	MS005467
Analysis ID:	AN005744
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Metabolites were measured with a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap Mass spectrometer (HRMS) coupled to a Dionex Ultimate 3000 UHPLC. The mass spectrometer was operated in full-scan, polarity-switching mode, with the spray voltage set to +4.5 kV/-3.5 kV, the heated capillary held at 320 °C, and the auxiliary gas heater held at 280 °C. The sheath gas flow was set to 55 units, the auxiliary gas flow was set to 15 units, and the sweep gas flow was set to 0 unit. HRMS data acquisition was performed in a range of m/z = 70–900, with the resolution set at 70,000, the AGC target at 1 × 106, and the maximum injection time (Max IT) at 120 ms. Metabolite identities were confirmed using two parameters: (1) precursor ion m/z was matched within 5 ppm of theoretical mass predicted by the chemical formula; (2) the retention time of metabolites was within 5% of the retention time of a purified standard run with the same chromatographic method. Chromatogram review and peak area integration were performed using the Thermo Fisher software Tracefinder 5.0 and the peak area for each detected metabolite was normalized against the total ion count (TIC) of that sample to correct any variations introduced from sample handling through instrument analysis. The normalized areas were used as variables for further statistical data analysis.
Ion Mode:	UNSPECIFIED