Abstract
The development of new methodology for the direct analysis of protein-protein interactions is of high interest, as protein complexes are involved in all cellular processes. If mass spectrometry is routinely used for protein analysis, it is still challenging to use this analytical tool for the analysis of protein-protein interactions. Most of the succesful studies in this field have been done using electrospray ionization, requiring time-consuming optimizations both on sample preparation and instrumental settings. MALDI mass spectrometry is a faster method for the analysis of proteins but is rarely used for the direct analysis of protein-protein interactions. The principal reason is the high tendency of non-covalent interactions to dissociate during the ionization step. Another issue is the detection of the intact complexes as single-charge pseudomolecular ions. Here, we present the analysis of intact protein complexes by MALDI mass spectrometry using the combination of chemical cross-linking and high-mass MALDI mass spectrometry. To circumvent the dissociation problem, a specific chemical cross-linking reaction is performed prior to the MS analysis, maintaining the integrity of the intact complex. To allow the detection of the complex, a new high-mass detection system is used, allowing sensitive analysis (nM range) in the 10–1200 kDa range. Different applications will be presented: Immunochemistry: Direct analysis of immuno-complexes between prion/antiprion; HAtag/antiHA; GSTtag/antiGST; BSA/AntiBSA in the 150–400 kDa range; analysis of epitope by competition assays, sandwich assays. Protein pathways: Direct analysis of complex stoichiometry for the complexes: AMPk, CDC42-SopE, thymidine K, ATP synthase, ferritin complex. Supershifting: The supershifting application consists in determining the presence of a subunit in a complex by MALDI high-mass mass spectrometry using monoclonal antibodies. An example of a supershifting experiment will be presented with the complexes formed by the fusion proteins GST-SopE and GST-CDC42 using a monoclonal antibody anti-GST.