The data was processed to identify lipids using Agilent’s lipid annotator software. Briefly, a series of five iterative MSMS were run and a list of MSMS confirmed lipids was obtained from the iterative MSMS using Agilent’s lipid annotator software. All the samples were then processed against the library using Agilent’s Profinder software. A total of 417 lipids were confidently identified based on this workflow. Each lipid was identified based on their MS, MS/MS and retention time assumption.
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Liquid Chromatography MS - negative - reverse-phase","Liquid Chromatography MS - positive - reverse-phase"],"chromatography_protocol":["The analysis was performed using an ultra-high pressure liquid chromatography (UHPLC) system (1290 Infinity II, Agilent Technologies) coupled to an Agilent quadrupole time-of-flight mass spectrometry (6545 QTOF-MS) with electrospray ionization (ESI) source. The UHPLC system consisted of a degasser, two binary pumps, temperature-controlled autosampler and column oven. MassHunter data acquisition software for the 6200 series, TOF/6500 series QTOF (version B.08.01) was used to control LC-MS acquisition. To assure the desired mass accuracy of recorded ions, continuous internal calibration was executed using signals from purine (m/z 121.0509 in positive ESImode and m/z 112.050873 in negative ESI mode) and hexakis (1H, 1H, 3H-tetrafluoropropoxy) phosphazine (m/z 922.0098 in positive ESI mode and m/z 980.016375 in negative ESI mode). Resolution of 12,000 FWHM (full width at half maximum) at m/z 121 and m/z 112 and 24,000 FWHM at m/z 922 and 980 was achieved. Reverse Phase liquid chromatography (RPLC) separation was obtained using Waters Xbridge C18 (3.5 µm, 2.1x150 mm) column and a guard column.
Positive ESI mode: The mobile phase A, water/methanol (60/40, v/v) and the mobile phase B 2-propanol/methanol (90/10, v/v), both with 10 mM ammonium acetate. Samples were analyzed using 20 µL/min flow rate at 55°C and the following gradient: 0-2 min 20% B; 2-4 min 30%B, 4-25 min 80%B, 25-35 min 85%B, 35-38 min 95%B, 38-41 min 95%B and 41.1-50 min 20%B Injection volume of 10 µL was used for all analyses. Mass spectrometry detection settings included: N2 drying gas temperature 200°C; N2 drying gas flow 10 L/min; nebulizer pressure 30 psig, Sheath gas temperature 300°C, sheath gas flow 12 L/min, capillary voltage 3500 V, and fragmentor voltage of 175 V. MS data was collected for m/z range of 200-1200 at the acquisition rate of 3 spectra/s in positive ESI mode.
Negative ESI mode: Binary solvent system consisted of 0.02% acetic acid in solvent A water/methanol (60/40, v/v) and solvent B 2-porpanol/methanol (90/10, v/v). Same gradient, column temperature and injection volume as positive method was used. Mass spectrometry detection was performed in negative ESI full scan mode with a mass range of 285 to 1200 m/z. The mass spectrometer source conditions consisted of a capillary voltage of 3000 V. Drying and sheath gas temperatures were set to 200 and 300°C and flow rates to 10 and 12 L/minutes, respectively. Nebulizer pressure was set to 40 psig and fragmentor voltage to 175 V. Data was acquired in centroid mode at the rate of 3 spectra per second in the extended dynamic range mode (2 GHz).
"],"publication":["Cholesterol dysregulation contributes to hematopoietic failure in parn-mutant zebrafish with implications for dyskeratosis congenita."],"submitter_affiliation":["CHEO Research Institute"],"submitter_name":["Sarada Ketharnathan"],"organism_part":["Kidney marrow"],"technology_type":["mass spectrometry assay"],"disease":[""],"extraction_protocol":["On the day of extraction, tissue homogenization was done by beating for 2 mins at 30Hz (bead beating was done twice) after adding 594 µL of acetonitrile. Samples were then transferred to 5mL Eppendorf tube and incubated with a 2:1 dichloromethane:water solution on ice for 10 minutes. The polar and non-polar phases were separated by centrifugation at 4000xg for 10 minutes at 1°C. The upper polar phase was dried using a refrigerated CentriVap Vacuum Concentrator at -4°C (LabConco Corporation). Dried samples were resuspended in 50µL methanol containing internal standard (Deuterated Ceramide LIPIDOMIX® Mass Spec Standard 330713X and SPLASH® LIPIDOMIX® Mass Spec Standard, 330707, from Avantipolar) (Supplemental Data 1, internal standard tab). After reconstitution the samples were vortexed well, centrifuged and then transferred to HPLC vial (with insert) for LC-MS analysis. Extraction blank was also prepared following the same protocol by using water instead of WKM tissue. 10 µL aliquots from each sample were pooled to prepare a quality control (QC) sample, which was then used to condition the column and served as QC throughout the run. 5 µl extracts were injected into the HPLC system.
Positive ESI mode: The mobile phase A, water/methanol (60/40, v/v) and the mobile phase B 2-propanol/methanol (90/10, v/v), both with 10 mM ammonium acetate. Samples were analyzed using 20 µL/min flow rate at 55°C and the following gradient: 0-2 min 20% B; 2-4 min 30%B, 4-25 min 80%B, 25-35 min 85%B, 35-38 min 95%B, 38-41 min 95%B and 41.1-50 min 20%B Injection volume of 10 µL was used for all analyses. Mass spectrometry detection settings included: N2 drying gas temperature 200°C; N2 drying gas flow 10 L/min; nebulizer pressure 30 psig, Sheath gas temperature 300°C, sheath gas flow 12 L/min, capillary voltage 3500 V, and fragmentor voltage of 175 V. MS data was collected for m/z range of 200-1200 at the acquisition rate of 3 spectra/s in positive ESI mode.
Negative ESI mode: Binary solvent system consisted of 0.02% acetic acid in solvent A water/methanol (60/40, v/v) and solvent B 2-porpanol/methanol (90/10, v/v). Same gradient, column temperature and injection volume as positive method was used. Mass spectrometry detection was performed in negative ESI full scan mode with a mass range of 285 to 1200 m/z. The mass spectrometer source conditions consisted of a capillary voltage of 3000 V. Drying and sheath gas temperatures were set to 200 and 300°C and flow rates to 10 and 12 L/minutes, respectively. Nebulizer pressure was set to 40 psig and fragmentor voltage to 175 V. Data was acquired in centroid mode at the rate of 3 spectra per second in the extended dynamic range mode (2 GHz).
"],"organism":["Danio rerio"],"full_dataset_link":["https://www.ebi.ac.uk/metabolights/MTBLS15863"],"author":["Sarada Ketharnathan. CHEO Research Institute. sarada.biotech@gmail.com.","Jason Berman. CHEO Research Institute. JBerman@cheo.on.ca."],"data_transformation_protocol":["Data quality for all the samples was performed using Agilent Mass Hunter TOF Qualitative Analysis software (version B.10.00). The overall QC repeatability and sample TIC were manually verified for any redundancies. The reproducibility of spiked internal standard was verified in all QCs and samples separately (Supplemental Data 1). The data was processed to identify lipids using Agilent’s lipid annotator software. Briefly, a series of five iterative MSMS were run and a list of MSMS confirmed lipids was obtained from the iterative MSMS using Agilent’s lipid annotator software. All the samples were then processed against the library using Agilent’s Profinder software. A total of 417 lipids were confidently identified based on this workflow. Each lipid was identified based on their MS, MS/MS and retention time assumption.
Peak values were normalized using extraction blanks. Hierarchical clustering analysis was performed on log10 transformed and pareto-scaled data using Metaboanalyst 6.0.
"],"study_factor":["Genotype"],"submitter_email":["sarada.biotech@gmail.com"],"sample_collection_protocol":["Whole kidney marrow tissue from 4 parn+/+ and 4 parn-/- fish at 9 months of age were dissociated and snap frozen.
"],"omics_type":["Metabolomics"],"study_design":["ultra-performance liquid chromatography-mass spectrometry","Metabolomics","dyskeratosis congenita","untargeted analysis","Kidney marrow","Danio rerio","Agilent 1290 Infinity II UHPLC","Lipidomics","ultra high-performance liquid chromatograph","inherited bone marrow failure","Agilent 6545 Q-TOF","MassHunter Qualitative Analysis"],"curator_keywords":["ultra-performance liquid chromatography-mass spectrometry","Metabolomics","dyskeratosis congenita","untargeted analysis","Kidney marrow","Danio rerio","Agilent 1290 Infinity II UHPLC","Lipidomics","ultra high-performance liquid chromatograph","inherited bone marrow failure","Agilent 6545 Q-TOF","MassHunter Qualitative Analysis"],"mass_spectrometry_protocol":["The analysis was performed using an ultra-high pressure liquid chromatography (UHPLC) system (1290 Infinity II, Agilent Technologies) coupled to an Agilent quadrupole time-of-flight mass spectrometry (6545 QTOF-MS) with electrospray ionization (ESI) source. The UHPLC system consisted of a degasser, two binary pumps, temperature-controlled autosampler and column oven. MassHunter data acquisition software for the 6200 series, TOF/6500 series QTOF (version B.08.01) was used to control LC-MS acquisition. To assure the desired mass accuracy of recorded ions, continuous internal calibration was executed using signals from purine (m/z 121.0509 in positive ESImode and m/z 112.050873 in negative ESI mode) and hexakis (1H, 1H, 3H-tetrafluoropropoxy) phosphazine (m/z 922.0098 in positive ESI mode and m/z 980.016375 in negative ESI mode). Resolution of 12,000 FWHM (full width at half maximum) at m/z 121 and m/z 112 and 24,000 FWHM at m/z 922 and 980 was achieved. Reverse Phase liquid chromatography (RPLC) separation was obtained using Waters Xbridge C18 (3.5 µm, 2.1x150 mm) column and a guard column.
Positive ESI mode: The mobile phase A, water/methanol (60/40, v/v) and the mobile phase B 2-propanol/methanol (90/10, v/v), both with 10 mM ammonium acetate. Samples were analyzed using 20 µL/min flow rate at 55°C and the following gradient: 0-2 min 20% B; 2-4 min 30%B, 4-25 min 80%B, 25-35 min 85%B, 35-38 min 95%B, 38-41 min 95%B and 41.1-50 min 20%B Injection volume of 10 µL was used for all analyses. Mass spectrometry detection settings included: N2 drying gas temperature 200°C; N2 drying gas flow 10 L/min; nebulizer pressure 30 psig, Sheath gas temperature 300°C, sheath gas flow 12 L/min, capillary voltage 3500 V, and fragmentor voltage of 175 V. MS data was collected for m/z range of 200-1200 at the acquisition rate of 3 spectra/s in positive ESI mode.
Negative ESI mode: Binary solvent system consisted of 0.02% acetic acid in solvent A water/methanol (60/40, v/v) and solvent B 2-porpanol/methanol (90/10, v/v). Same gradient, column temperature and injection volume as positive method was used. Mass spectrometry detection was performed in negative ESI full scan mode with a mass range of 285 to 1200 m/z. The mass spectrometer source conditions consisted of a capillary voltage of 3000 V. Drying and sheath gas temperatures were set to 200 and 300°C and flow rates to 10 and 12 L/minutes, respectively. Nebulizer pressure was set to 40 psig and fragmentor voltage to 175 V. Data was acquired in centroid mode at the rate of 3 spectra per second in the extended dynamic range mode (2 GHz).
"],"additional_accession":[]},"is_claimable":false,"name":"Cholesterol dysregulation contributes to hematopoietic failure in parn-mutant zebrafish with implications for dyskeratosis congenita","description":"Germline mutations in PARN cause dyskeratosis congenita (DC), an inherited bone marrow failure syndrome characterized by multilineage cytopenia and progression to myeloid disease. The mechanisms underlying this malignant transformation have not been fully elucidated. Here we show, using a loss-of-function CRISPR-Cas9 zebrafish parn mutant, hematopoietic stem and progenitor (HSPC) expansion and reduced mature hematopoietic cells across various lineages. Cholesterol biosynthesis was transcriptionally activated; a finding recapitulated in patient-derived iPSCs with PARN mutations. Inhibiting cholesterol biosynthesis rescued HSPC expansion and anemia in parn mutants. Concurrent myeloid differentiation therapy or Wnt inhibition reduced HSPCs to varying degrees. We further demonstrate a role for activated Erk signaling in impairing erythrocyte differentiation and show robust rescue with trametinib treatment. Overall, our findings uncover unrecognized roles for Parn in regulating cholesterol metabolism and Erk signaling and provide preclinical evidence for targeting these pathways to restore hematopoiesis in DC.","dates":{"publication":"2026-09-29","submission":"2026-09-29"},"accession":"MTBLS15863","cross_references":{"MetaboLights":["MTBLC140947"],"ChEBI":["CHEBI:140947"]}}