No abstract

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 , 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.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein–protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

The mechanism of the CuAAC reaction has been investigated by electrospray ionization mass spectrometry (ESI-MS) using a combination of the neutral reactant approach and the ion-tagging strategy. Under these conditions, for the first time, putative dinuclear copper intermediates were fished out and characterized by ESI(+)-MS/MS. New insight into the CuAAC reaction mechanisms is provided and a catalytic cycle is proposed.

The number of publications in the field of chemical cross-linking/mass spectrometry (MS) for deriving protein 3D structures and for probing protein/protein interactions has largely increased during the last years. MS analysis of the large cross-linking data sets requires an automated data analysis by dedicated software tools, but applying scoring procedures with statistical methods does not eliminate the fundamental problems of a misassignment of cross-linked products. In fact, we have observed a significant rate of misassigned cross-links in a number of publications, mainly due to the presence of isobaric cross-linked species, an incomplete fragmentation of cross-linked products, and low-mass accuracy fragment ion data. These false assignments will eventually lead to wrong conclusions on the structural information derived from chemical cross-linking/MS experiments. In this contribution, we examine the most common sources for misassigning cross-linked products. We propose and discuss rational criteria and suggest five guidelines that might be followed for a reliable and unambiguous identification of cross-links, independent of the software used for data analysis. In the interest of the cross-linking/MS approach, it should be ensured that only high-quality data enter the structural biology literature. Clearly, there is an urgent need to define common standards for data analysis and reporting formats of cross-linked products.