Journal: Cell Discovery

Article Title: TGM2-mediated serotonylation of GPX4 confers ferroptosis resistance to promote gastric tumorigenesis

doi: 10.1038/s41421-026-00885-6

Figure Lengend Snippet: a TGM2-dependent transamidation signals in recombinant GPX4 (monitored via monodansylcadaverine fluorescence) that were abolished by either cystamine inhibition or 5-HT competition. b Schematic illustration of the TGM2-dependent transamidation of glutamine residues in substrate proteins. c MS/MS spectra of the serotonylated GPX4 peptide (GPX4ser: Q55, Q77) derived from GPX4 (in vitro). The b ion refers to the N-terminal portion of the peptide, and the y ion indicates to the C-terminal portion. d SILAC experimental workflow for GPX4 serotonylation detection by LC-MS/MS using 5-HT and 5-HT- d 4 . TGM2 catalyzes the transamidation of glutamine residues in the GPX4 protein. e Chromatographic overlay of 5-HT and 5-HT- d 4 , demonstrating nearly identical retention times or 45.03 min and 45.05 min, respectively (top). MS analysis of 5-HT and 5-HT- d 4 revealed distinct isotopic peaks. Mass spectra of Light, with m/z of 727.36 and Heavy, with m/z of 729.37 m/z for 5-HT and 5-HT- d 4 , respectively, illustrating the clear separation between the light and heavy isotopic labels and confirming the use of deuterium-labeled serotonin for dual validation (bottom). f Tandem MS/MS spectrum of the peak at m/z 727.36 (5-HT Light) showing the fragmentation pattern of GPX4 at the serotonylated site, confirming the presence of 5-HT modification (top). Tandem MS/MS spectrum of the peak at m/z 729.37 (5-HT- d 4 Heavy) showing the corresponding fragmentation pattern, confirming the site-specific serotonylation of GPX4 (bottom). g Immunoblot and streptavidin pull-down analysis of Flag-GPX4 with various mutations (Q54A, Q77A, and Q2A) in HEK293T cells treated with 5-PT, confirming the loss of serotonylation at specific sites. Quantification is shown on the right (mean ± SEM; ordinary one-way ANOVA, n = 3).

Article Snippet: Each 50 μL reaction mixture contained 1× 50 mM HEPES reaction buffer (1× protease inhibitor), 5 mM 5-HT- d 4 (MCE, HY-B1473S) or 5 mM 5-HT combined with 5 mM CaCl 2 , 1.25 μg TGM2, and GPX4 recombinant protein (50 μg) and was established on ice.

Techniques: Recombinant, Fluorescence, Inhibition, Tandem Mass Spectroscopy, Derivative Assay, In Vitro, Multiplex sample analysis, Liquid Chromatography with Mass Spectroscopy, Labeling, Biomarker Discovery, Modification, Western Blot