CANDLE supporting metabolomics datasets
Description
Supporting targeted metabolomics datasets for "CANDLE syndrome 20S proteasome mutations cause altered neuronal development related to dysregulated polyamine synthesis"
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Metabolomics Sample Extraction All CO and biofluid samples were directly immersed in 0.4 mL of ice-cold methanol. NPCs and neurons in culture were first washed with 1 mL of 0.9 % NaCl, prior to the addition of 0.4 mL ice-cold methanol. Cells were then scraped to suspend all debris and transferred to microtube for further processing. To each sample 0.4 mL of water and 0.4 mL of chloroform were added. Samples were shaken for 30 minutes under refrigeration and centrifuged at 16k xg for 20 min. 400 µL of the top (aqueous) layer was collected and diluted as necessary in 50 % methanol in water for LC-MS/MS analysis. Metabolomics (Liquid Chromatography Tandem Mass Spectrometry) Tributylamine was purchased from Millipore Sigma. LCMS grade water, methanol, isopropanol, formic acid, and acetic acid were purchased through Fisher Scientific. Aqueous metabolites were analyzed using two analytical methods with opposing ionization polarities (Groveman et al., 2023; McCloskey et al., 2015). Both methodologies utilized a LD40 XR UHPLC (Shimadzu Co.) system for separation and a 6500+ QTrap mass spectrometer (AB Sciex Pte. Ltd.) for detection. In negative mode methodology, samples were separated with a Waters™ Atlantis T3 column (100Å, 3 µm, 3 mm X 100 mm) and eluted using a binary gradient from 5 mM tributylamine, 5 mM acetic acid in 2% isopropanol, 5% methanol, 93% water (v/v) to 100% isopropanol over 5 minutes. Two distinct multiple reaction monitoring (MRM) ion pairs in negative mode were used for each metabolite where available. In positive mode methodology, samples were separated using a Phenomenex Kinetex F5 column (100 Å, 2.6 µm, 100 x 2.1 mm) and eluted with a gradient from 0.1 % formic acid in water to 0.1 % formic acid in acetonitrile over 5 minutes. Biocrates Targeted Metabolomics Assay A targeted metabolomic assessment was carried out following the manufacturer’s protocol using Biocrates’ MxP® Quant 500 kit, in organoids (20 µl). For 6 organoids, 300 µl of 85% Ethanol and 15% PBS was homogenized in 2 ml tubes from the Precellys Lysing Kit, soft tissue homogenizing CK14 (Bertin, Rockville, MD). The tubes were then homogenized for 2 cycles of 20 s at 6500 rpm with 30 s interval between the homogenization steps using the Precellys 24 between 0-4oC. Afterwards the tubes were centrifuged with a benchtop centrifuge and the solution was transferred to a separate 1.5 ml Eppendorf tube, where they were centrifuged for 10 mins at 17,000-x g in the cold room and the samples were stored at -80oC until analysis. Samples were run on a Nexera HPLC system (Shimadzu) coupled to a 6500+ QTRAP® mass spectrometer (AB Sciex) with an electrospray ionization source as previously described (13). Data were quantified using WebIDQ™ software and normalized to internal quality controls. Any metabolite that had greater than 30% above limit of detection (LOD) was excluded, while those with less than 30% LOD had their values imputed as 1/5 the lowest concentration.
Institutions
- National Institute of Allergy and Infectious Diseases