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We present a concise workflow to enhance the mass spectrometric detection of cross-linked peptides by introducing sequential digestion and the cross-link identification software Xi. Sequential digestion enhances peptide detection by shortening long tryptic peptides while avoiding over-digestion. We demonstrate our simple 12-fraction protocol for cross-linked multi-protein complexes and cell lysates, quantitative analysis, and high-density cross-linking, without requiring specific cross-linker features. This overall approach reveals dynamic protein-protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.

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