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

Abstract 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.