High-efficiency cross-linker TDS enables low-input in vivo structural and interactomic analysis.

In vivo chemical cross-linking mass spectrometry (XL-MS) is powerful for capturing protein-protein interactions and dynamics in native contexts, but its high sample requirement (typically E7-E8 cells) hinders application to rare cell populations. To address this, we developed a low-input in vivo XL-MS strategy, that synergizes a miniaturized, transfer-free one-pot workflow with TDS (trehalose disuccinimidyl ester) cross-linker. TDS is highly biocompatible and membrane-permeable, enabling efficient in vivo cross-linking with minimal cellular perturbation. Crucially, TDS is MS-cleavable; upon fragmentation, it generates diagnostic fragment ions that linearize the data retrieval, dramatically simplifying identification and enhancing sensitivity compared to the quadratic complexity inherent to non-cleavable cross-linkers. Furthermore, its inherent LC-MS-compatibility permits retention throughout processing without interference, obviating intermediate cleanup steps and rendering the workflow seamless for low-input applications. Notably, this strategy delineates a dense interaction network comprising 273 proteins linked by 1055 unique cross-links from just 1E3 cells. This performance effectively bridges the gap between low-input constraints and high-resolution structural analysis of core cellular machinery. We exemplify this capability by probing the conformational dynamics of histone H3.2 from low-input sample. Collectively, this methodology provides a robust platform for deciphering interactome architectures from trace biological specimens.