Integrated Proteomic and PTM-Proteomic Profiling Reveals Dose-Dependent Responses to S-Adenosylmethionine in Hepatocellular Carcinoma Cells

Abstract Hepatocellular carcinoma (HCC) remains a major cause of cancer-related mortality. S-Adenosylmethionine (SAM) is a central methyl donor with context-dependent effects on HCC growth, but the associated proteomic responses remain incompletely characterized. We performed integrated data-independent acquisition (DIA)-based proteomic, phosphoproteomic, and methylproteomic analyses of Huh7 cells treated with 0, 20, or 200 μM SAM. In Huh7 cells, 20 μM SAM increased CCK-8-based cell growth and migration, whereas 200 μM SAM reduced growth and increased cell death. AML12 cells showed no comparable reduction in CCK-8 signal under the tested conditions, although this comparison cannot establish tumor selectivity. We identified 5,197 proteins, 1,522 phosphorylation sites from 795 proteins, and 322 methylation sites from 262 proteins. Proteins carrying SAM-associated methylated-peptide changes were enriched in RNA splicing and translation-related processes. High-concentration SAM was associated with decreased ribosome-related protein abundance, metabolic remodeling, moderately increased ROS-associated fluorescence, and stress- and apoptosis-related phosphosite changes. Parent-protein adjustment retained selected relative PTM-site signals but attenuated others, indicating contributions from both PTM-associated and protein-abundance changes. Exploratory TCGA-LIHC analyses provided gene-level clinical context for selected candidates. These findings provide a multilayer proteomic resource for investigating concentration-dependent SAM responses and prioritizing proteins and PTM sites for functional validation.