A Detergent-Free Sample Preparation Method Enabling Desalting-Free Tandem PTM Enrichment

Using K-GG antibody beads, CaTiO₃, and ZIC-HILIC, DFSP-PTM facilitated the direct enrichment of modified peptides, yielding approximately 6,000 ubiquitinated peptides, 30,000 phosphopeptides, and 800 intact glycopeptides (1% glycan FDR) from 200 μg HeLa proteins. The DFSP-PTM workflow further identified ∼8000 proteins, 8072 localized ubiquitination sites (DIA-NN score >0.9), 18,584 localized phosphosites and 2117 glycosylation sites in IFNγ-stimulated samples, demonstrating high reproducibility (Pearson correlation >0.8).

Furthermore, we successfully applied this workflow to investigate <i>in vivo</i> molecular events in vesicular stomatitis virus (VSV)-infected mice. In summary, DFSP-PTM provides a rapid, robust, and universally applicable platform for deep, multidimensional PTM profiling.