iProXProteomicsXiuling LiHomo SapiensMus Musculushttp://www.iprox.org/page/project.html?id=IPX0009561000lixiuling@dicp.ac.cnDalian Institute of Chemical PhysicsiProXSerial multi-omic analysis of proteome, phosphoproteome, and glycoproteome is pivotal for elucidating drug mechanisms, discovering biomarkers, and identifying therapeutic targets. However, simultaneous multi-level post-translational modifications (PTMs) analysis via parallel processing is hampered by laborious, time-consuming procedures and inconsistent reproducibility. We present an integrated Multi-level PTMs-Proteomic Enrichment platform (MuPPE), enabling sequential glycoproteome, phosphoproteome, and proteome analysis from single biological samples. It combines protein aggregation capture with on-bead digestion and tandem enrichment, achieving superior reproducibility (CV 12.3% vs 17.6% conventional methods) while reducing processing time by 87.5% (4 hours vs 32 hours). MuPPE also enhances coverage, identifying more serum glycopeptides and brain phosphopeptides than other platforms. Applied to aging mouse cohorts, the platform uncovers tissue-specific PTMs remodeling and brain barrier dysfunction. For arsenic mechanisms of action, MuPPE reveals drug-induced PTMs crosstalk between glycosylation and phosphorylation-driven pathway regulation. MuPPE offers a transformative tool for advancing multi-omics insights across precision medicine and disease research.A versatile platform for sequential glyco-, phospho-, and proteomics with multi-PTMs integration.Dong Xuefang X, Xiong Fangfang F, Du Guangzhu G, Yang Yunfei Y, Chen Cheng C, Cui Yun Y, Ding Xinlian X, Li Xiuling X, Shen Yidong Y, Liang Xinmiao XfalseMass spectrometry data for proteomics and PTM proteomicsMass spectrometry data for proteomics and PTM proteomicsSat Jan 11 00:00:00 GMT 2025PXD05970110090960641605919