iProXProteomicsHongqiang QinHomo Sapienshttp://www.iprox.org/page/project.html?id=IPX0017205000qinhq@dlut.edu.cnState Key Laboratory of Fine Chemicals, Department of Pharmaceutical Engineering, School of Chemical Engineering, Dalian University of TechnologyiProXLysine dimethylation (Kme2) is a critical post-translational modification (PTM), while its comprehensive analysis remains challenging due to low stoichiometry and lack of specific antibodies. Here, we developed an antibody-free proteomics strategy combining selective fluorine-mediated oxidation with reversible hydrazide enrichment. In this approach, Kme2 residues were oxidized to aldehyde, covalently captured by hydrazine-functionalized beads, and reversibly released for MS analysis. This approach achieved high specificity, identifying 293 and 284 site-specific Kme2 events in HeLa and A549 cells, respectively. Based on the good reproducibility, quantitative analysis enabled precise characterization of Kme2 sites sensitive to the methylation inhibitor Adox. Applied to breast cancer models, we found that the Kme2 events on cytoskeletal and metabolic adaptation were enhanced in metastatic MDA-MB-231 cells, while the Kme2 events were increased on translational machinery components. Thus, the antibody-free platform provides a robust tool for Kme2 mapping and methylation-targeted drug discovery, offering insights into epigenetic regulation in cancer progression.Antibody-Free Profiling of Lysine Dimethylation via Chemoselective Oxidation-Coupled Reversible Enrichment.Li Qi Q, Zhang Zhe Z, Huang Anliang A, Zhang Zhuo Z, Yue Xuyang X, Yin Peiyuan P, Chen Xuwei X, Ye Mingliang M, Qin Hongqiang HfalseAntibody-Free Profiling of Lysine Dimethylation via Chemoselective OxidationCoupled Reversible EnrichmentLysine dimethylation (Kme2) is a critical post-translational modification (PTM), while its comprehensive analysis remains challenging due to low stoichiometry and lack of pan-specific antibodies. Here, we developed an antibody-free proteomics strategy combining selective fluorine-mediated oxidation with reversible hydrazide enrichment. In this approach, Kme2 residues were oxidized to aldehyde, covalently captured by hydrazine-functionalized beads, and reversibly released for MS analysis. This approach achieved high specificity and reproducibility, identifying 293 and 284 site-specific Kme2 events in HeLa and A549 cells, respectively. Based on the excellent reproducibility, quantitative analysis enabled precise characterization of Kme2 sites sensitive to the pan-methylation inhibitor Adox. Applied to breast cancer models, the approach revealed cell-type-specific Kme2 signatures: metastatic MDA-MB-231 cells exhibited cytoskeletal and metabolic adaptation Kme2 events, while proliferative MCF-7 cells prioritized translational machinery components. Our antibody-free platform provides a robust tool for Kme2 mapping and methylation-targeted drug discovery, offering insights into epigenetic regulation in cancer progression.Mon May 18 00:00:00 GMT+01:00 2026PXD078472960642234804