Using Covalent Labeling and Mass Spectrometry To Study Protein Binding Sites of Amyloid Inhibiting Molecules

摘要

src="http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancham/2017/ancham.2017.89.issue-21/acs.analchem.7b02915/20171101/images/medium/ac-2017-02915h_0005.gif">Amyloid aggregates are associated with several debilitating diseases, and there are numerous efforts to develop small molecule treatments against these diseases. One challenge associated with these efforts is determining protein binding site information for potential therapeutics because amyloid-forming proteins rapidly form oligomers and aggregates, making traditional protein structural analysis techniques challenging. Using β-2-microglobulin (β2m) as a model amyloid-forming protein along with two recently identified small molecule amyloid inhibitors (i.e., rifamycin SV and doxycycline), we demonstrate that covalent labeling and mass spectrometry (MS) can be used to map small-molecule binding sites for a rapidly aggregating protein. Specifically, three different covalent labeling reagents, namely diethylpyrocarbonate, 2,3-butanedione, and the reagent pair EDC/GEE, are used together to pinpoint the binding sites of rifamycin SV, doxycycline, and another molecule, suramin, which binds but does not inhibit Cu(II)-induced β2m amyloid formation. The labeling results reveal binding sites that are consistent with the known effects of these molecules on β2m amyloid formation and are in general agreement with molecular docking results. We expect that this combined covalent labeling approach will be applicable to other protein/small molecule systems that are difficult to study by traditional means.