Department of Pharmaceutical Chemistry and Bioanalytics, Martin-Luther University Halle-Wittenberg, Germany
Chemical cross-linking/mass spectrometry (XLMS) has emerged as a powerful tool for the 3D-structure analysis of proteins and protein complexes and is becoming increasingly popular in structural biology.
We have developed and successfully applied MS-cleavable cross-linkers and integrated workflows to perform XLMS at different levels, ranging from isolated protein and protein assemblies to highly complex protein mixtures, such as cell lysates and intact cells. Our protocols can be conducted within one week and are based on the commercially available MS-cleavable cross-linker disuccinimidyl dibutyric urea (DSBU). The workflows 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 (www.StavroX.com) that allows a fully automated analysis to deliver insights into protein interaction networks and protein conformations on a proteome-wide scale. We demonstrate the successful application of our workflow for Drosophila embryo extracts as well as intact E.coli cells and human embryonic kidney cells. Principles of modern cross-linkers and recent applications of XLMS will be discussed.
Ospite: Rita Grandori