New Tools for Proteome-Wide Cross-Linking Mass Spectrometry

This project deals with the establishment of methods and techniques to investigate protein-protein interaction networks within living cells. Such complex networks are essential for all biochemical processes within a cell and their regulation. To deepen knowledge on protein crosstalk is therefore one of the most important questions to answer in basic research. To visualize such a crosstalk, chemical reagents called cross-linkers are used to selectively connect and stabilize proteins at positions in close proximity. After that, proteins can be isolated and cut into pieces using an enzyme. With the help of a mass- spectrometer the proteins can be identified, and the exact position of their interaction can be localized. This works by detection of a fingerprint resulting from the cut protein parts. Unfortunately, the chemical reaction efficiency of such cross-linkers is quite low, hampering a direct detection of those within the mass-spectrometer. To overcome this issue, we will design, develop and test novel cross-linker reagents with improved reactivity. Reactive sidechains attached to the cross-linkers will furthermore enable a selective enrichment. By combination of these strategies, we aim to obtain purified samples in sufficient amounts, in which the cross-link detection works effectively. In the next step, the recorded data has to be analyzed with the help of an algorithm. To do so, we already developed software that can identify more cross-links while being less prone to errors compared to competitors. During this project, the software will be further advanced, and a machine-learning algorithm will improve the exact localization of the cross-link on the protein. The functionality will be additionally expanded to several types of cross-linkers. In a final project step, we will apply the established holistic workflow to investigate differences in protein-protein interactions between areas of the cell, where the genetic material is condensed vs. better accessible. This research work will significantly deepen the knowledge about how genetic material is read within cells.