Proteomics and biomolecular mass spectometry of hemostatic processes

The binding between individual proteins that are involved in a hemostatic process, like blood coagulation, is typically part of a complex protein-protein interaction network. Taking maximum advantage of the versatility of the nano-LC LTQ Orbitrap XL ETD mass spectrometer, several studies have been initiated to unravel complex mechanisms involved in protein-protein interactions, storage of hemostatic proteins in the secretory organelles, and intracellular processing of proteins. An example of the last study involves immune tolerance induction against coagulation factor VIII (FVIII). This study required the identification of FVIII peptides that are presented on dendritic cells via the MHC class II proteins. For this purpose, immature monocyte-derived dendritic cells were incubated with FVIII to allow for its uptake and intracellular processing. After extraction of the MHC class II molecules from the cells, the FVIII peptides bound to these molecules were identified employing mass spectrometry. The study has now provided an unbiased identification of donor dependent T-cell epitopes of FVIII.

Another example involves a so-called chemical foot printing approach in which we scan for solvent exposed regions on the coagulations factors VIII and IX. This method provides insight into the functional regions of the coagulation factors as well as changes therein during their activations. We will further implement mass spectrometry technologies that will allow for the identification of differentially expressed proteins, or changes in modifications thereof in treated versus non-treated cells. These technologies will be particularly suitable to unravel protein signaling networks in cellular systems.

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