2026-10-10T09:51:35Z https://api.figshare.com/v2/oai

oai:figshare.com:article/33986539 2026-09-24T15:30:23Z category_1 category_4 category_21 category_734 category_135 portal_63 item_type_3 month_year_09_2026

A Detergent-Free Sample Preparation Method Enabling Desalting-Free Tandem PTM Enrichment Zhan-Peng Lin (25104625) Xinyan Gao (3569468) Chengwen Wen (11889675) Jia-Xin Wang (138350) Wanyue Luo (25104628) Chong Tu (25104631) Xuekun Wang (1885210) Xiaohua Jiang (177009) Chuan-Qi Zhong (550216) Biophysics Biochemistry Biotechnology Biological Sciences not elsewhere classified Computational Biology yielding approximately 6 vesicular stomatitis virus universally applicable platform native conditions followed initial tryptic cleavage entirely eliminating detergents demonstrating high reproducibility 9 ), 18 800 intact glycopeptides 584 localized phosphosites 2117 glycosylation sites gg antibody beads free sample preparation consuming desalting steps 000 ubiquitinated peptides multidimensional ptm profiling ptm profiling sample loss modified peptides free multi 000 phosphopeptides ptm provides ptm facilitated vivo </ using k unique two translational modifications successfully applied subsequent time stimulated samples second digestion ptms ). powerful tool pearson correlation nn score molecular events inevitably lead highly streamlined glycan fdr directly compatible direct enrichment based methods analyzing post 8 ). 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 <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. 2026-09-24T00:00:00Z Dataset Dataset 10.1021/acs.jproteome.6c00349.s001 https://figshare.com/articles/dataset/A_Detergent-Free_Sample_Preparation_Method_Enabling_Desalting-Free_Tandem_PTM_Enrichment/33986539 CC BY-NC 4.0