iProXProteomicsChuanqi ZhongHomo Sapienshttp://www.iprox.org/page/project.html?id=IPX0012058000zhongcq@xmu.edu.cnXiamen UniversityiProXfalseA detergent-free and desalting-free sample preparation method for tandem-enrichment of muiltiple PTMsMass spectrometry (MS)-based proteomics requires efficient sample preparation for post-translational modification (PTM) analysis. Here, we present a ​detergent- and desalting-free method (DFSP)​​ enabling tandem enrichment of ubiquitination, phosphorylation, and glycosylation. DFSP employs a two-step digestion: initial tryptic cleavage under native conditions followed by heat-induced denaturation (95°C, 10 min), achieving proteolytic efficiency comparable to detergent-based methods (75,974 peptides identified). By eliminating desalting steps, DFSP directly enriches PTM peptides using K-GG antibody beads, CaTiO3, and ZIC-HILIC, yielding ​10,130 ubiquitinated peptides, ​47,057 phosphopeptides, and ​700 glycopeptides​ from 500 μg HeLa proteins. Integrated with diaPASEF-MS and computational platforms (FragPipe/DIA-NN), DFSP revealed IFNγ-induced regulation of 18,584 phosphosites and 2,117 glycosites, with high reproducibility (Pearson correlation >0.8). This streamlined workflow reduces processing time and sample loss, offering a robust tool for multi-PTM profiling in complex biological systems.Mon Apr 27 00:00:00 GMT+01:00 2026PXD0776789606