One‐Step Enrichment and Quantitative Analysis of In Vivo Protein Complexes via Dimethylpiperidine Cross‐Linker DPST

In vivo cross-linking mass spectrometry (XL-MS) enables the proteome-wide characterization of protein complexes in living cells. However, most XL-MS methods face significant sample loss during enrichment, limiting their applications to limited-quantity samples, and suffer from poor reproducibility (20%-40%), hindering precise quantification. To overcome these challenges, we developed a novel membrane-permeable cross-linker, 2,6-dimethylpiperidine disuccinimidyl tridecanoate (DPST), in which the dimethylpiperidinyl group enables one-step enrichment of cross-linked peptides via tandem mass tags (TMTs) antibody approach, eliminating sample loss from multi-step processes and allowing analysis from as few as 1E4 cells. DPST also allows the light and heavy isotopic labeling of cross-linked samples at the cellular level, which reduces inaccuracies from multi-step preparations. This generates reporter ions for precise MS2 quantification, improving the signal-to-noise ratio without increasing spectral complexity. Using DPST, we analyzed cross-links in primary neurons from single fetal mice and quantified the transient and weak interactions in dynamic liquid-liquid phase separation (LLPS) environments. Additionally, DPST's design supports multiple isotopic labeling configurations (e.g., 6-plex, 10-plex). Therefore, DPST provides a scalable and robust tool for in vivo XL-MS-based qualitative and quantitative analysis of living cells, even with limited sample quantities.