ABSTRACTWe present a cross-linking/mass spectrometry (XLMS) workflow for performing proteome-wide cross-linking analyses within one week. The workflow is based on the commercially available MS-cleavable cross-linker disuccinimidyl dibutyric urea (DSBU) and can be employed by every lab having access to a mass spectrometer with tandem MS capabilities. We provide an updated version 2.0 of the freeware software tool MeroX, available at www.StavroX.com, that allows conducting fully automated and reliable studies delivering insights into protein-protein interaction networks and protein conformations at the proteome level. We exemplify our optimized workflow for mapping protein-protein interaction networks in Drosophila melanogaster embryos on a system-wide level. From cross-linked Drosophila embryo extracts, we detected 18,037 cross-link spectrum matches corresponding to 5,129 unique cross-linked residues in biological triplicate experiments at 5% FDR (3,098 at 1% FDR). Among these, 1,237 interprotein cross-linking sites were identified that contain valuable information on protein-protein interactions. The remaining 3,892 intra-protein cross-links yield information on conformational changes of proteins in their cellular environment.
The Application of an Emerging Technique for Protein–Protein Interaction Interface Mapping: The Combination of Photo-Initiated Cross-Linking Protein Nanoprobes with Mass Spectrometry
