A Simple Cross-Linking/Mass Spectrometry Workflow for Studying System-wide Protein Interactions

We present a cross-linking/mass spectrometry workflow for performing proteome-wide cross-linking analyses within 1 week. The workflow is based on the commercially available mass spectrometry-cleavable cross-linker disuccinimidyl dibutyric urea and can be employed by every lab having access to a mass spectrometer with tandem mass spectrometry capabilities. We provide an updated version 2.0 of the freeware software tool MeroX, available at www.StavroX.com, that allows us to conduct 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 29931 cross-link spectrum matches corresponding to 7436 unique cross-linked residues in biological triplicate experiments at a 1% false discovery rate. Among these, 1611 interprotein cross-linking sites were identified and yielded valuable information about protein–protein interactions. The 5825 remaining intraprotein cross-links yield information about the conformational landscape of proteins in their cellular environment.ISSN:1520-6882ISSN:0003-270