MetaboLights

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bolights/studies/public/MTBLS15875Metabolites were annotated using the EI-Maven software (Elucidata, https://www.elucidata.io/el-maven) with an offset of ± 15 ppm. The intensity of each target was normalized to the internal standards (positive/negative standards for metabolites) and protein concentration.  The identification of lipid molecular species was performed manually considering their characteristic fragments and/or neutral losses, retention time and exact m/z. Data processing and analysis were done through the combined use of SeeMS® software and an in-house Excel-based macro established within our laboratory.  Using EI-Maven, the peak areas of individual lipid species were extracted from the MS data.. Each peak integration was carefully inspected to ensure the accuracy of peak detection and area determination. The area ratio for each lipid was normalized to the peak area of its respective internal standard.  The concentration of lipid molecular species and fatty acids was estimated by multiplying the area ratio by the concentration of the corresponding internal standard. Final concentrations were reported as nmol/mg of protein.MetaboLightsPublicLiquid Chromatography MS - alternating - hilicLiquid Chromatography MS - negative - reverse-phaseLiquid Chromatography MS - positive - reverse-phaseWater-soluble metabolites were analysed by LC-MS/MS using a Q-Exactive Plus quadrupole-Orbitrap mass spectrometer (Thermo) interfaced with an ultra high-performance liquid chromatography (Ultimate 3000, Thermo). The analytical gradient was performed using an SeQuant ZIC-pHILIC column (5 µm, 2.1 mm x 150 mm, Millipore Sigma) with solvent A (20 mM ammonium carbonate + 0.1% ammonium hydroxide in water) and solvent B (acetonitrile), as previously described [29]. Briefly, the samples (3 µL) were injected into the column at 25 °C, and the analytical gradient lasted 30 minutes, as follows: linear gradient from 80% to 20% within 20 minutes before returning to 80% in 0.5 min. and appended with a 7.5-minute equilibration step. The flow rate was kept at 150 µL/min.  Total lipid extracts were loaded into a BEH® (UPLC® C18 column, 1.7 µm, 2.1 mm i.d. x 100 mm) with a flow rate of 0.2 mL min−1 and the oven temperature maintained at 40 °C. Mobile phase A of the reverse-phase LC consisted of water/acetonitrile (60:40), while mobile phase B composed of isopropanol/acetonitrile/water (88:10:2). Mobile phases A and B included 10 mM ammonium formiate and 0.1% formic acid for experiments performed in negative or positive ionization modes. LC gradient analysis conditions were carried out exactly as previously described (Chaves-Filho et al., 2019).Loss of SPTSSA Impairs Mitochondrial Function and Neuronal Differentiation in SH-SY5Y Neuroblastoma Cells.German Cancer Research CenterAdriano de Britto Chaves FilhoWhole Organismmass spectrometry assayCells were harvested with 700 µL methanol/water (4:1) containing internal standards (Supplementary Tables 3 and 4) and transferred to Eppendorf tubes. Polar metabolites and lipids were extracted using MTBE, as previously described with modifications (Matyash et al., 2008). Briefly, 1 mL of tert-butyl methyl ether (MTBE) was added to each sample, which was then vortexed for 30 seconds and agitated at 1000 rpm at 20 ºC for 1 h in a thermomixer. Subsequently, 300 μL of ultrapure water was added and the samples were kept in an ice-cold bath for 10 minutes. Phase separation was achieved by centrifugation at 10,000 × g at 4°C for 10 min, the lipid-containing organic fraction was collected in a glass vial, dried under a nitrogen stream and reconstituted in 80 μL of isopropanol. The aqueous fraction was vacuum-dried using a Speedvac and resuspended in 80 μL of H2O/ACN (25/75). The residual pellet was reserved for protein quantification by the BCA assay.Homo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS15875Adriano Chaves-Filho. German Cancer Research Center. adriano.debrittochavesfilho@dkfz-heidelberg.de.Aysu Kök. German Cancer Research Center. aysubasakkok@gmail.com.Roger Sandhoff. German Cancer Research Center. r.sandhoff@dkfz.de.Raw files were converted into mzML files before data analysis.SPTSSAadriano.debrittochavesfilho@dkfz-heidelberg.deSH-SY5Y cell lines (both SPTSSA KO and control versions) were plated into 6-well culture plates with 400,000 cells per well, using five replicate wells for each condition. After a 48-hour incubation, the culture medium was removed, the cells were rinsed with PBS, and then rapidly frozen using liquid nitrogen. MetabolomicsMetabolomicsMitochondrial dysfunctiontargeted analysisSerine palmitoyltransferaseuntargeted analysisHomo sapiensThermo Scientific Dionex Ultimate 3000 HPLC systemNeuronal DifferentiationThermo Scientific Q Exactive PlusWhole OrganismMetabolomicsMitochondrial dysfunctiontargeted analysisSerine palmitoyltransferaseuntargeted analysisHomo sapiensThermo Scientific Dionex Ultimate 3000 HPLC systemNeuronal DifferentiationThermo Scientific Q Exactive PlusWhole OrganismPolar metabolites were analysed with MS in ESI positive/negative mode with ddMS2. The full scan at 70k resolution (69-1000 m/z scan range, 1e6 AGC-Target, 50 ms maximum Injection Time (maxIT)) was followed by a ddMS2 at 17.5k resolution (1 x 105 AGC target, 20 ms maxIT, 1 loop count, 2 s to 10 s apex trigger, 2 x 103 minimum AGC target, 20 s dynamic exclusion). The HESI source parameters were set as 40 sheath gas flow rate, 15 auxiliary gas flow rate, 1 sweep gas flow rate, spray voltage: 3.0 kV in positive mode, 3.1 kV in negative mode, 320 °C capillary temperature, and the heater temperature of auxiliary gas was 120 °C. Total lipid extracts were analysed by LC-MS/MS using the same instruments described for analysis of water-soluble metabolites. The MS was operated in both positive and negative ionization modes, and the scan range was set at a mass-to-charge ratio of 350-1600 Da and 350-1300 Da, respectively. The mass spectrometry was operated using sheath gas flow rate: 30; auxiliary gas flow rate: 10; sweep gas flow rate: 0; spray voltage: 3.6 kV (positive)/2.5 kV (negative); capillary temperature: 320 °C; S-lens RF level: 55.0; auxiliary gas heater temperature: 120 °C. Data acquisition on MS was carried out using the following settings: mass resolution = 70,000 at m/z 200, Automatic Gain Control (AGC) = 1 x 106; and Injection Time (IT) = 50 ms. Data for lipid molecular species identification and quantification was obtained by data dependent acquisition (DDA) using the following settings: mass resolution = 17,500 at m/z 200, AGC = 1x105; IT = 50 ms; isolation window = 1.2 m/z; collision energy (CE) = 20/40/60; dynamic exclusion = 10s; and Minimum AGC target to trigger MS/MS = 2 x 103.  falseLoss of SPTSSA Impairs Mitochondrial Function and Neuronal Differentiation in SH-SY5Y Neuroblastoma CellsSphingolipids are bioactive membrane and signaling lipids that not only shape cellular architecture but also orchestrate a network of interconnected metabolic pathways. Their core building blocks, sphingoid bases, are synthesized by the serine palmitoyltransferase (SPT) complex. At its core, the SPT complex contains two large subunits (SPTLC1 with either SPTLC2 or SPTLC3) and one small subunit (SPTSSA or SPTSSB). Thereby it establishes the ganglioside composition of the aging brain. To investigate the functional impact of altered sphingolipid composition on cell physiology, we generated SPTSSA knockout (KO) SH-SY5Y neuroblastoma cell lines still expressing SPTSSB and characterized them through multiomics approaches. SPTSSA KO led to increase in the ratio of C20/C18-sphingosine containing ceramides. Multiomics analysis revealed significant mitochondrial dysfunction, with upregulation of electron transport chain complexes I, III and V but downregulation of complex IV. These changes led to reduced ATP levels, alternation in NAD+/NADH and GSH/GSSG ratios. Elevated L-lactic acid and glyceraldehyde 3-phosphate levels indicated a metabolic shift toward glycolysis. Most critically, upon B27/all-trans retinoic acid (ATRA) treatment, SPTSSA KO cells had impaired neuronal differentiation capacity and gradually died over time. Overall, alterations in SPT complex composition through SPTSSA deletion altered cellular proteome and metabolome. These findings demonstrate that fine-tuning and proper ratio of C18- and C20-sphingosine containing sphingolipids are essential for mitochondrial homeostasis and successful neuronal differentiation in SH-SY5Y neuroblastoma cells.2026-10-092026-09-30MTBLS15875