MetaboLightsapplication/xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15161/m_MTBLS15161_LC-MS_positive_reverse-phase_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15161/s_MTBLS15161.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15161/a_MTBLS15161_LC-MS_positive_reverse-phase.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15161/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15161/FILES/RAW_FILES/HepG2_GFP_5days_1.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15161/FILES/RAW_FILES/HepG2_FL_5days_1.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15161/FILES/RAW_FILES/HepG2_d8_5days_2.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15161/FILES/RAW_FILES/HepG2_GFP_5days_3.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15161/FILES/RAW_FILES/HepG2_FL_5days_3.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15161/FILES/RAW_FILES/HepG2_d8_5days_3.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15161/FILES/RAW_FILES/HepG2_FL_5days_2.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15161/FILES/RAW_FILES/HepG2_d8_5days_1.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15161/FILES/RAW_FILES/HepG2_GFP_5days_2.d.zipprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15161<p>Metabolites were detected using a targeted LC-MS/MS multiple reaction monitoring (MRM) method. Features were defined a priori by the acquisition method as compound-specific precursor/product ion transitions with associated collision energies and retention time windows. Raw data were acquired using Agilent MassHunter software and processed in Agilent MassHunter Quantitative Analysis software for peak integration and quantification. Metabolite annotations were assigned based on the predefined MRM transition list, and where applicable, retention time matching to authentic standards/internal standards. No untargeted feature detection, spectral library search, or external metabolite database annotation pipeline was used.</p>MetaboLightsPublicLiquid Chromatography MS - positive - reverse-phase<p>Samples were resuspended in 0.1% formic acid, clarified by centrifugation, and 8 microliters were injected into an LC–MS Agilent 6490 triple quadrupole (QQQ) system equipped with a Phenomenex Luna 100 Å 75 x 2.0 mm C18 column. A multi-reaction monitoring (MRM) approach was used to quantify dozens of metabolites (see Supplementary Table S2 for MRM parameters). The mobile-phase gradient was delivered at a flow rate of 0.4 mL/min over a 13.01-min run. The LC method began at 100% solvent A (0.1% formic acid) and 0% solvent B (95% acetonitrile, which was maintained until 2.00 min. Solvent B was then increased to 100% by 7.00 min and held at 100% B until 9.50 min. The gradient was returned to initial conditions of 100% A and 0% B at 9.51 min and maintained through 13.00 min for column re-equilibration. At 13.01 min, the method switched to 0% A and 100% B.</p>The GNMT N-terminus Couples Folate Feedback to Methyl-donor Homeostasis.Albert Einstein College of MedicineIsaac KrazHep-G2 cellmass spectrometry assay<p>Metabolites from cultured cells were extracted using an ice-cold extraction buffer composed of 40% acetonitrile, 40% methanol, 20% water, and 0.1% formic acid, supplemented with stable isotope-labeled SAM, SAH, glycine, and sarcosine as internal standards for spike-in normalization. The cell–extraction buffer mixture was scraped into microcentrifuge tubes, and insoluble debris was pelleted by centrifugation (18,000 × g, 4 °C, 10 min). The supernatant was transferred to fresh tubes, flash-frozen in liquid nitrogen, and dried overnight in a SpeedVac.</p>Homo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS15161David Shechter. Albert Einstein College of Medicine. david.shechter@einsteinmed.edu.Isaac Kraz. Albert Einstein College of Medicine. isaac.krasnopolsky@einsteinmed.edu.<p>For a given metabolite using the Agilent MassHunter software, raw peaks were integrated and normalized to peak area of the spiked in ISTD (e.g. 3D-SAM), thus yielding an ISTD ratio. These values were then normalized to the original cell number counted for each condition and an absolute, scaled cellular concentration was determined.</p>Biological replicateisaac.krasnopolsky@einsteinmed.edu<p>Metabolites from cultured HepG2 cells after 5 days of growth following lentiviral overexpression of target protein. </p>MetabolomicsMetabolomicsHep-G2 celltargeted analysisAgilent softwarenegative control roleAgilent 6490 Triple QuadrupoleHomo sapiensMetabolismAgilent 1290 Infinity HPLCmetabolic dysfunction-associated steatotic liver diseaseexperimental sampleMetabolomicsHep-G2 celltargeted analysisnegative control roleAgilent softwareAgilent 6490 Triple QuadrupoleHomo sapiensMetabolismAgilent 1290 Infinity HPLCmetabolic dysfunction-associated steatotic liver diseaseexperimental sample<p>Mass spectrometric detection was performed on an Agilent G6490A triple quadrupole mass spectrometer equipped with an XESI source and operated in positive-ion MRM mode. Source parameters were as follows: drying gas temperature, 200 °C; drying gas flow, 14 L/min; nebulizer pressure, 20 psi; sheath gas temperature, 250 °C; sheath gas flow, 11 L/min; capillary voltage, 3000 V; and charging voltage, 1500 V. MRM transitions were acquired at low resolution for both Q1 and Q3 with a dwell time of 200 ms, fixed fragmentor voltage of 380 V, cell acceleration voltage of 5 V, delta EMV of 0 V, and high- and low-pressure funnel RF values of 150 and 60 V, respectively. Collision energies ranged from 10 to 80 eV. Monitored precursor ions ranged from m/z 75.9 to 613.1, and product ions ranged from m/z 30.2 to 483.0</p>G6PspermidineGSSG5-methyltetrahydrofolateglutathionedimethylglycinepyruvatecholinemethioninealpha ketoglutarateSAHMTAbetaineSAMfalseThe GNMT N-terminus Couples Folate Feedback to Methyl-donor HomeostasisMaintenance of S-adenosylmethionine (SAM) homeostasis is essential for methylation of biomolecules, nucleotide and polyamine synthesis, and redox balance. While all methyltransferases consume SAM, only a subset of highly tissue specific methyltransferases regulates methylation potential. Among them, glycine N-methyltransferase (GNMT) is enriched in the liver and its dysregulated activity has been linked to compromised liver function. GNMT is inhibited by the methyl carrier 5-methyltetrahydrofolate (5mTHF), suggesting a negative-feedback mechanism regulating its activity. Here, we identify the GNMT N-terminal tail, and specifically phosphorylation at serine 9 (S9ph), as a regulatory modification linking folate-dependent feedback inhibition to SAM homeostasis. Structural and biochemical analyses and molecular dynamics simulations revealed that the N-terminal tail is required for catalytic turnover of SAM and for 5mTHF binding. Phosphoproteomic analysis showed that GNMT S9ph is abundant in mouse liver and further enriched in aged mice. Consistent with loss of folate-dependent negative feedback, both distal N-terminal truncation (residues 1-8) and a phosphomimetic substitution abolished 5mTHF binding while maintaining catalytic activity. In hepatocyte cell lines lacking endogenous GNMT, lentiviral overexpression of constitutively active GNMT mutants depleted SAM, increased SAH, disrupted protein methylation, impaired growth, and induced transcriptional responses consistent with methyl-donor stress. Together, these findings identify the GNMT N-terminus as a tunable phosphoregulatory domain that dynamically regulates GNMT activity and cellular methylation potential.2026-08-182026-07-27MTBLS15161MTBLC15414MTBLC178058MTBLC17509MTBLC18608MTBLC17858MTBLC16856MTBLC15354MTBLC17724MTBLC64558MTBLC17665MTBLC15361MTBLC17750MTBLC57834CHEBI:15414CHEBI:178058CHEBI:17509CHEBI:18608CHEBI:17858CHEBI:16856CHEBI:15354CHEBI:17724CHEBI:64558CHEBI:17665CHEBI:15361CHEBI:17750CHEBI:57834