Metabolites were annotated using a targeted LC-MS workflow based on accurate mass and chromatographic retention time. Peak annotation and integration were performed using El-Maven (Elucidata). Ornithine, putrescine, spermidine and spermine were quantified using stable isotope-labelled internal standards (13C5-ornithine, 13C4-putrescine, 13C4-spermidine and D8-spermine). Acetylated polyamines were monitored as targeted analytes and are reported as relative/raw peak abundances.
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Liquid Chromatography MS - positive - reversed-phase-c18"],"chromatography_protocol":["Chromatography – C8-D1A cells
Separation was performed on a Thermo Scientific Vanquish LC system equipped with a Waters ACQUITY UPLC HSS C18 column (1.7 µm, 2.1 × 100 mm). 10 μL of the extract was loaded. Mobile phase A consisted of water containing 0.2% acetic acid and mobile phase B consisted of acetonitrile containing 0.2% acetic acid. Flow rate was 250 µL/min and column temperature was maintained at 30°C. The gradient started at 20% B, increased linearly to 85% B between 2 and 10 min, was maintained until 17 min, and returned to starting conditions at 18 min. Total run time was 22 min.
Chromatography – ACM, NCM, plasma, cortex and CSF
Separation was performed on a Thermo Scientific Vanquish LC system coupled to a Waters ACQUITY UPLC HSS T3 VanGuard pre-column (100 Å, 1.8 µm, 2.1 × 5 mm). 50 μL of the extract was loaded. Mobile phase A consisted of water containing 0.2% acetic acid and mobile phase B consisted of acetonitrile containing 0.2% acetic acid. Column temperature was maintained at 25°C. A rapid gradient elution method was applied with a total runtime of 5.53 min. A gradient for the separation of modified polyamines was applied as follows: Flow rate started at 1.8 mL/min with 5% B while running from column to the waste to prevent reagents from entering the mass spectrometer, changing from 5 to 35% B from 0.67 to 1 min. From 1 to 1.1 min the flow increased linearly from 1.8 mL/min to 0.3 mL/min and the % B increased to 36%, then the flow from the column was redirected to the MS. From 1.1 to 3 min a linear increase to 85% B was carried out and remained at 85% B until 4.7 min. From 4.7 min to 4.73 min the flow switched to the waste again, and increased to 1.8 mL/min. From 4.73 to 4.86 min %B was linearly decreased to 5% B and kept there for equilibration. The method stopped at 5.53 min. The column temperature remained constant at 25 °C.
"],"publication":["ATP13A4 gates extracellular polyamine levels to control excitatory synaptogenesis. 10.1101/2025.04.04.25325117."],"submitter_name":["Sarah van Veen"],"submitter_affiliation":["Katholieke Universiteit Leuven"],"organism_part":["blank","blood plasma","cell culture medium","cerebrospinal fluid","Cerebral Cortex","astrocyte cell line C8-D1A"],"technology_type":["mass spectrometry assay"],"disease":[""],"extraction_protocol":["Extraction – C8-D1A cells
Cellular metabolites were extracted using 150 µL ice-cold 6% trichloroacetic acid (TCA). The cell lysate was collected using a cell scraper and incubated on ice for 30 min. Next, the lysate was centrifuged at 20,000 x g for 20 min at 4 °C. The supernatant was collected, flash-frozen in liquid nitrogen, and stored at -80 °C for future analysis. 1,6-Hexanediamine (Sigma, Cat H11696) was added as an internal standard to correct for signal drift. Correction was only performed when doing so improved replicate reproducibility. To 100 μL of supernatant, 900 μL of 100 mM sodium carbonate buffer (pH 9.3) (Sigma, Cat S7795-500g) was added. Then, 25 μL of isobutyl chloroformate (Sigma, Cat 177989-25G) was introduced, and the mixture was incubated at 35 °C for 30 min. Afterward, 800 μL of the reaction mixture was transferred to a 2 mL Eppendorf tube, followed by the addition of 1 mL of diethyl ether (Sigma, Cat 309966-1L). The mixture was vigorously vortexed and allowed to sit at 25 °C for 15 min. Subsequently, 900 μL of the upper phase was collected in a new Eppendorf tube and dried using a vacuum centrifuge. The dried extract was then dissolved in 125 μL of 50% acetonitrile (LC–MS grade; VWR, Cat 83640.320) in water containing 0.1% formic acid (VWR, Cat 84865.260) and transferred to an MS vial.
Extraction – ACM/NCM, mouse brain tissue and plasma samples
For ACM and plasma samples, 50 μL of ice-cold TCA extraction buffer was added to 50 μL of sample. For cerebrospinal fluid (CSF), extraction was performed using a matched-volume approach to account for limited and variable sample input. Each CSF sample was combined with an equal volume of ice-cold 6% TCA extraction buffer. Due to the limited volume obtained per animal, a subset of CSF samples was pooled, prior to extraction, to ensure sufficient material for LC-MS/MS analysis. Pooling was performed across animals of the same genotype and sex, and pooled samples were processed identically to individual samples. In figures, individual samples are represented as filled symbols, whereas pooled samples are indicated as open symbols. For cortical brain tissue (15-30 mg), 250 μL of ice-cold TCA extraction buffer was used, and samples were homogenized in Ribolyser tubes to ensure thorough extraction. For absolute quantification of polyamine levels, internal standards were included in the extraction buffer. The standards used were 13C5 ornithine (Cat CLM-4724-H-PK), 13C4 putrescine (Cat CLM-6574-PK), 13C4 spermidine (Cat CLM-9435-PK) and D8 spermine (Cat DLM-9262-PK) from Cambridge Isotope Laboratories, Inc.
To 40 μL of supernatant, 360 μL of 100 mM sodium carbonate buffer (pH 9.3) was added. Then, 10 μL of isobutyl chloroformate (Sigma) was introduced, and the mixture was incubated at 35 °C for 30 min. Afterward, the reaction mixture was transferred to an MS vial.
"],"organism":["blank sample","Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/metabolights/MTBLS14724"],"author":["Sarah van Veen. Katholieke Universiteit Leuven. sarah.vanveen@kuleuven.be.","Peter Vangheluwe. Katholieke Universiteit Leuven. peter.vangheluwe@kuleuven.be."],"data_transformation_protocol":["Raw LC-MS data were processed using El-Maven (Elucidata). Peak detection and integration were performed using extracted ion chromatograms. Ornithine, putrescine, spermidine and spermine were corrected using their corresponding stable isotope-labelled internal standards (13C5-ornithine, 13C4-putrescine, 13C4-spermidine and D8-spermine) for ACM, plasma, CSF and tissue samples; or 1,6-hexanediamine for C8-D1A cell extracts. Acetylated polyamines lacking compound-specific internal standards were not corrected and are reported as integrated raw peak abundances.
"],"study_factor":["Sex","Genotype"],"submitter_email":["sarah.vanveen@kuleuven.be"],"sample_collection_protocol":["Sample collection – C8-D1A cell extracts (files labeled MCF001037)
Mouse C8-D1A astrocyte cells were cultured under standard conditions. Cells were harvested at the experimental endpoint, washed with 0.9% NaCl and immediately processed for metabolite extraction.
Sample collection – Astrocyte-conditioned medium
Samples consisted of astrocyte-conditioned medium (ACM) (files labeled MCF002042) and non-conditioned medium (NCM) (files labeled MCF002322). Primary mouse/rat astrocytes were cultured and conditioned medium was collected following incubation in minimal medium. ACM was clarified by filtration and concentrated prior to metabolomic analysis. Samples were stored at −80°C until extraction and LC-MS analysis.
Sample collection – Mouse tissues and biofluids
Samples consisted of cortical brain tissue (files labeled MCF001869 and MCF002051), plasma (files labeled MCF001869 and MCF002051) and cerebrospinal fluid (CSF) (files labeled MCF002386) and MCF002051) collected from two-month-old Atp13a4 WT and KO mice. Plasma was isolated from whole blood by centrifugation. CSF was collected from the cisterna magna and cortical tissue was rapidly dissected following euthanasia. All samples were snap-frozen and stored at −80°C until metabolomic analysis.
"],"omics_type":["Metabolomics"],"study_design":["astrocyte-conditioned medium","Metabolomics","Mus musculus","blank","cell extract","targeted analysis","Cerebral Cortex","experimental sample","non-conditioned medium control","polyamine","targeted metabolomic assay","blank sample","blood plasma","cell culture medium","cerebrospinal fluid","liquid chromatography mass spectrometry assay","experimental blank","Thermo Scientific Vanquish LC System","Thermo Scientific Q Exactive Orbitrap Focus","astrocyte cell line C8-D1A"],"curator_keywords":["astrocyte-conditioned medium","Metabolomics","Mus musculus","blank","cell extract","targeted analysis","Cerebral Cortex","experimental sample","non-conditioned medium control","polyamine","targeted metabolomic assay","blank sample","blood plasma","cell culture medium","cerebrospinal fluid","liquid chromatography mass spectrometry assay","experimental blank","Thermo Scientific Q Exactive Orbitrap Focus","Thermo Scientific Vanquish LC System","astrocyte cell line C8-D1A"],"mass_spectrometry_protocol":["Mass spectrometry – C8-D1A cells (files labeled MCF001037)
Mass spectrometric analysis was performed using a Thermo Scientific Q Exactive Orbitrap Focus mass spectrometer equipped with a heated electrospray ionization source operating in positive ion mode. Full scan data were acquired over an m/z range of 70–750. Sheath gas was set to 25, auxiliary gas to 10, ion transfer tube temperature to 320°C and vaporizer temperature to 310°C.
Mass spectrometry – ACM (files labeled MCF002042), NCM (files labeled MCF002322), plasma (files labeled MCF001869 and MCF002051), cortex (files labeled MCF001869 and MCF002051) and CSF (files labeled MCF002386)
The mass spectrometer operated in full scan (range 70.0000-1050.0000) and positive mode using Sheath gas at 25, Aux Gas at 10. Ion Transfer tube was heated at 320 °C and Vaporizer temperature was set at 310 °C. The data analyses were performed by integrating the peak areas (El-Maven - Elucidata).
Note for CSF samples (files labeled MCF002386): You will notice there are two sets of datafiles, one with the prefix mass350-1050_MCF002386 and one without.
The one with provided results for spermine and its internal standard, acetylspermidine and acetylspermine, and was obtained with a slightly different mass filter mentioned to increase sensitivity for these compounds as signal were unusually low.
The other provided results for all other compounds and internal standards.
"],"additional_accession":[]},"is_claimable":false,"name":"Targeted polyamine metabolomics of ATP13A4-deficient cellular and mouse models","description":"ATP13A4 is a polyamine transporter enriched in astrocytes that regulates extracellular polyamine homeostasis. This study contains targeted LC-MS/MS metabolomics data from ATP13A4-deficient cellular and mouse models, including C8-D1A astrocytic cells, astrocyte-conditioned medium, mouse cortex, plasma and cerebrospinal fluid. Polyamines and acetylated polyamine derivatives were quantified following isobutyl chloroformate derivatization using Orbitrap mass spectrometry. The dataset supports the manuscript 'ATP13A4 gates extracellular polyamine levels to control excitatory synaptogenesis' and investigates how ATP13A4 deficiency alters intracellular and extracellular polyamine distribution.
","dates":{"publication":"2026-06-10","submission":"2026-06-09"},"accession":"MTBLS14724","cross_references":{}}