Journal: Molecular & Cellular Proteomics : MCP
Article Title: Jolkinolide B Activates Mitophagy to Exhibit Antipancreatic Cancer Activity and Alleviate Cognitive Deficits in Alzheimer's Disease
doi: 10.1016/j.mcpro.2025.101060
Figure Lengend Snippet: Mass spectrometry directly identifies TOM40 as a covalent binding target of JB. A , workflow for the direct identification of JB targets using mass spectrometry. MIA PaCa-2 cells were treated with 25 μM JB or DMSO for 24 h. Iodoacetamide (IAA) served as an alkylating reagent for cysteine; the purple star represents the JB molecule. B , schematic illustrating the combined analysis of forward and reverse strategies used to screen potential JB targets. XCorr represents the reliability score of the spectrum; Identified both refers to peptides with carbamidomethyl modification in the DMSO group and JB modification in the JB treatment group. TMT: tandem mass tag; DEPs: differentially expressed proteins; GO: gene ontology. C , cellular thermal shift assay (CETSA) was conducted to measure the binding affinity of JB to TOM40 in MIA PaCa-2 cells. Data represent mean ± s.e.m. from at least three independent experiments. D , recombinant TOM40 protein was preincubated with JB (0, 5, 50, or 500 μM) for 2 h, then incubated with 5 μM HJB-Cy7 for competitive binding. E , recombinant TOM40 protein was incubated with or without JB (50 or 500 μM) for 2 h, and 5-IAF was used to label cysteine residues on TOM40. F – G WT TOM40 protein and mutant C74S protein were incubated with HJB-Cy7 or JB. Labeled proteins were scanned using the ChemiDoc MP imaging system, and silver staining was performed as a loading control. 5-IAF, 5-iodoacetamidofluorescein; DEP, differentially expressed protein; DMSO, dimethyl sulfoxide; HJB, 17-hydroxy-jolkinolide B; JB, jolkinolide B.
Article Snippet: Samples were vortexed with loading buffer, subjected to 12% SDS-PAGE, and visualized using a ChemiDoc MP scanner (Bio-Rad).
Techniques: Mass Spectrometry, Binding Assay, Modification, Thermal Shift Assay, Recombinant, Incubation, Mutagenesis, Labeling, Imaging, Silver Staining, Control
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