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J Proteome Res. 2019 Jun 7;18(6):2545-2558. doi: 10.1021/acs.jproteome.9b00085. Epub 2019 May 24.

Optimized Cross-Linking Mass Spectrometry for in Situ Interaction Proteomics.

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Department of Pediatrics, Pharmacology, and Physiology & Biophysics, Weill Cornell Medical College , Cornell University , New York , New York 10065 , United States.


Recent development of mass spectrometer cleavable protein cross-linkers and algorithms for their spectral identification now permits large-scale cross-linking mass spectrometry (XL-MS). Here, we optimized the use of cleavable disuccinimidyl sulfoxide (DSSO) cross-linker for labeling native protein complexes in live human cells. We applied a generalized linear mixture model to calibrate cross-link peptide-spectra matching (CSM) scores to control the sensitivity and specificity of large-scale XL-MS. Using specific CSM score thresholds to control the false discovery rate, we found that higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) can both be effective for large-scale XL-MS protein interaction mapping. We found that the coverage of protein-protein interaction maps is significantly improved through the use of multiple proteases. In addition, the use of focused sample-specific search databases can be used to improve the specificity of cross-linked peptide spectral matching. Application of this approach to human chromatin labeled in live cells recapitulated known and revealed new protein interactions of nucleosomes and other chromatin-associated complexes in situ. This optimized approach for mapping native protein interactions should be useful for a wide range of biological problems.


BSA; chromatin; cross-linking; database search; false positive discovery; mass spectrometry; protein‚ąíprotein interactions; proteomics; target-decoy strategy

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