Optimized cross-linking mass spectrometry for in situ interaction proteomics Journal Article
| Authors: | Ser, Z.; Cifani, P.; Kentsis, A. |
| Article Title: | Optimized cross-linking mass spectrometry for in situ interaction proteomics |
| Abstract: | 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. © 2019 American Chemical Society. |
| Keywords: | mass spectrometry; proteomics; chromatin; bsa; cross-linking; protein-protein interactions; database search; false positive discovery; target-decoy strategy |
| Journal Title: | Journal of Proteome Research |
| Volume: | 18 |
| Issue: | 6 |
| ISSN: | 1535-3893 |
| Publisher: | American Chemical Society |
| Date Published: | 2019-06-07 |
| Start Page: | 2545 |
| End Page: | 2558 |
| Language: | English |
| DOI: | 10.1021/acs.jproteome.9b00085 |
| PUBMED: | 31083951 |
| PROVIDER: | scopus |
| PMCID: | PMC7473601 |
| DOI/URL: | |
| Notes: | Source: Scopus |
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