Disordered Motifs and Domains in Cell Control - October 11-15, 2014

Disordered Motifs and Domains in Cell Control

Poster Session II

44-POS Board 20 Mass Spectrometry Combined with Chemical Probing of Flexible Proteins Tim Verschueren , Frank Sobott. Biomolecular & Analytical Mass Spectrometry, Chemistry Department, University of Antwerp, Belgium Mass spectrometry can be used in combination with chemical probing techniques to characterize the flexible regions of proteins. Often used methods for protein characterization, such as x-ray crystallography and electron microscopy, are not always suited for the study of flexible (disordered) or highly dynamic proteins. We propose a combination of covalent solution labelling which targets the exposed, solvent accessible surface of proteins and complexes, with subsequent analysis by mass spectrometry (digestion followed by LC-MS/MS). The use of different reagents, time-titration and varying reaction conditions results in differences in probing patterns. The comparison of the extent, rate and location of modifications between the denatured and native state of a protein can tell us something about which regions are structured, as well as about the solvent accessibility of the surface area and by extension the flexibility of a protein. Selective modification of amino acids, e.g. histidines with diethylpyrocarbonate or lysines with dimethylamine borane, as well as type 0 crosslinkers induces only a limited number of modifications which ensures the structure integrity and avoids complex data. We also explore chemical as well as fast laser-induced (FPOP = Fast Photochemical Oxidation of Proteins) hydroxylation of the accessible protein surface. Optimization of these reactions as well as the combination with other MS-based techniques, such as ion mobility and top-down fragmentation, allow us to gain more insight into the structure and interactions of proteins. This makes mass spectrometry in combination with chemical probing particularly suited for the study of flexible proteins. We will highlight these different approaches using a panel of proteins with known degrees of structural flexibility and disordered regions (IDR).

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