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Reputable Mentor II
Reputable Mentor II
Liu F, Rijkers DTS, Post H, Heck AJR
Nat Methods. 2015 Sep 28. doi: 10.1038/nmeth.3603. [Epub ahead of print]
We describe an integrated workflow that robustly identifies cross-links from endogenous protein complexes in human cellular lysates. Our approach is based on the application of mass spectrometry (MS )-cleavable cross-linkers, sequential collision-induced dissociation (CID )–tandem MS (MS /MS ) and electron-transfer dissociation (ETD )-MS /MS acquisitions, and a dedicated search engine, XlinkX, which allows rapid cross-link identification against a complete human proteome database. This approach allowed us to detect 2,179 unique cross-links (1,665 intraprotein cross-links at a 5% false discovery rate (FDR ) and 514 interprotein cross-links at 1% FDR ) in HeLa cell lysates. We validated the confidence of our cross-linking results by using a target-decoy strategy and mapping the observed cross-link distances onto existing high-resolution structures. Our data provided new structural information about many protein assemblies and captured dynamic interactions of the ribosome in contact with different elongation factors.
University of Utrecht
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