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.