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High-Efficiency Cross-Linker TDS Enables Low-Input In Vivo Structural and Interactomic Analysis

ABSTRACT: 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 overcome this, we developed a novel low-input in vivo XL-MS strategy. This approach synergizes a miniaturized, transfer-free one-pot workflow with a custom-designed cross-linker, trehalose disuccinimidyl ester (TDS). 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 reporter ions that linearize the data retrieval process, dramatically simplifying identification and boosting sensitivity compared to the quadratic complexity of analyzing non-cleavable cross-linkers. Furthermore, its inherent MS-compatibility allows it to remain in the sample throughout processing without interference, eliminating cleanup steps that cause sample loss. This integrated strategy robustly captures interactome and structural information from as few as 1,000 HeLa cells, identifying a network of 273 proteins connected by 1,055 unique cross-links, and extends the detection limit to 100 cells.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Beirong Zhang;Yu Xia;Yi Liu

PROVIDER: PXD077718 | JPOST Repository | Sun Jun 28 00:00:00 GMT+01:00 2026

REPOSITORIES: jPOST

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			Action	DRS
	1E4_4_2.raw	Raw
	1E4_4_3.raw	Raw
	1_1.raw	Raw
	1_6.raw	Raw
	2_1_250.raw	Raw

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