Proteomics

Dataset Information

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Optimized cross-linking mass spectrometry for in situ interaction proteomics


ABSTRACT: ecent 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 density and coverage of protein-protein interaction maps can be significantly improved through the use of multiple proteases. In addition, the use of 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.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo sapiens  

TISSUE(S): Tissue Not Applicable To Dataset

DISEASE(S): Not Available

SUBMITTER: Alex Kentsis  

LAB HEAD: Alex Kentsis

PROVIDER: PXD010796 | Pride | 2019-04-03

REPOSITORIES: pride

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Publications

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

Ser Zheng Z   Cifani Paolo P   Kentsis Alex A  

Journal of proteome research 20190524 6


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-M  ...[more]

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