To cleave or not to cleave: a systemic evaluation of DSS versus DSSO for cross-linking mass spectrometry analysis

Cross-linking mass spectrometry is a powerful method for structural analysis, but choosing between cleavable and non-cleavable cross-linkers remains challenging. We rigorously compared non-cleavable DSS with cleavable DSSO and found that DSS consistently yields more cross-link identifications from isolated protein complexes to bacterial lysates. The advantage of DSS diminishes assample complexity increases. At the highest complexity tested—human cell lysate—the trend reverses, with DSSO out performingDSS. The superior performance of DSS in less complex samples islikely explained by its longer and more flexible spacer arm, which interrogates a spatial volume >40% larger than that of DSSO. Forboth cross-linkers, the number of identified cross-links decreases as the search space expands, but more steeply for DSS. This sharper decline arises from DSS cross-links producing slightlylower fragment ion coverage, not from the absence of signature ions that could reduce search space. Fragment ion coverage is key to interactome mapping: when coverage reaches 85% or above, identification sensitivity hardly decreases as the search space expands, regardless of the cross-linker used. In summary, we
recommend DSS for samples no more complex than bacterial lysates. For interactome mapping of mammalian cells, although DSSO outperforms DSS, neither achieves deep interactome coverage

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