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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