biostudies-literatureUnknown17(1)Dong XSerial 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 enhaNature communications948https://www.ebi.ac.uk/biostudies/studies/S-EPMC12852849biostudies-literatureA versatile platform for sequential glyco-, phospho-, and proteomics with multi-PTMs integration.PMC12852849Li XDing XYang YDong XLiang XCui YDu GChen CShen YXiong FfalseA versatile platform for sequential glyco-, phospho-, and proteomics with multi-PTMs integration.Serial 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 enha2026-01-01T00:00:00Z2026 Jan2026-06-10T09:05:36.73Z2026-06-10T03:11:36.297ZS-EPMC128528494160591910.1038/s41467-025-68270-7