High Mass MALDI-ToF MS for the examination of proteins and protein-protein interactions

The detection of large noncovalent protein complexes has been successfully realized in the Zenobi group by combining chemical cross-linking with high mass MALDI-ToF MS. Noncovalent interactions of complexes are thereby stabilized due to the reaction with a special cross-linker.
Basic but important knowledge concern the cross-linking step but is largely unknown (Fig. 1). Therefore we have started a project to determine reactivities of different reagents by working with several types of cross-linkers and investigate the kinetics of the reaction and its dependence on temperature. To learn more about protein-protein interactions, numerous systems, which can form a complex, are studied.

..... Fig. 1: Example for a cross-linking reaction of two proteins with DSG (Disuccinimidyl glutarate)

The instrumentations available in the laboratory allow the detection of stabilized complexes up to several MDa. Two MALDI-ToF mass spectrometers based on different type of detections are used: a Macromizer from the Comet corporation (Fig. 2 a) using a cryogenic detector and an Axima CFR (Kratos Analytical) fitted with a high mass detector from CovalX (Fig. 2 b) based on conversion of ions.