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

Abstract Mass spectrometry (MS)-based proteomics is a powerful tool for analyzing post-translational modifications (PTMs). However, conventional multiplexed PTM enrichment workflows typically require denaturing detergents and subsequent time-consuming desalting steps, which inevitably lead to sample loss. Here, we present a detergent-free sample preparation (DFSP) method for highly streamlined, desalting-free multi-PTM profiling. DFSP utilizes a unique two-step digestion strategy─an initial tryptic cleavage under native conditions followed by a second digestion after heat-induced denaturation─achieving proteolytic efficiency comparable to conventional detergent-based methods. By entirely eliminating detergents, DFSP allows generated peptides to be directly compatible with antibody- and bead-based enrichment strategies without prior intermediate desalting. 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 in vivo 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.