Probing protein-ligand interactions via solution phase hydrogen exchange mass spectrometry
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Date
2010Author
Esswein, Stefan Theo
Metadata
Abstract
Mass spectrometry is a versatile, sensitive and fast technique with which to probe
biophysical properties in biological systems and one of the most important analytical
tools in the multidisciplinary field of proteomics. The study of nativestate
proteins and their complexes in the gas-phase is well established and direct
infusion electrospray ionisation mass spectrometry (DI-ESI-MS) techniques are
becoming increasingly popular as a tool for screening and determining quantitative
information on protein-protein and protein-ligand interactions. However, complexes
retained by ESI-MS are not always representative of those in solution and
care must be taken in interpreting purely gas-phase results. This thesis details
modification and advancement of solution phase techniques devised by Gross et
al. utilising ESI-MS and Fitzgerald et al. applying matrix assisted laser desorption
ionisation (MALDI)-MS termed PLIMSTEX (protein-ligand interactions by
mass spectrometry, titration and hydrogen-deuterium-exchange)[1] and SUPREX
(Stability of unpurified proteins from rates of H/D exchange)[2] to quantify these
interactions with regards to high throughput analysis.
The first part of this thesis describes the different developmental stages of the devised
HPLC-front ends and their optimisation with myoglobin and insulin. The successfully
developed HPLC-front end in conjunction with PLIMSTEX and SUPREX
and ESI-MS then gets tested with self expressed and purified cyclophilin A(CypA)-
cyclosporin A (CsA) system, followed by a test screen with potential CypA binding
ligands. Dissociation constants (Kd’s) within one order of magnitude to reported
values are determined.
In the third part of this thesis the application of the devised ESI-SUPREX methodology
has been applied to anterior gradient 2 (AGr2) and the factor H complement
control proteins module 19-20 (fH19-20) exhibiting binding potential to a taggedhexapeptide
and a synthetic pentasaccharide, respectively, resulting in thermodynamical
data for these protein-ligand interactions. For the AGr2 system another
dimension of investigation has been added by temperature controlling the devised
ESI-SUPREX approach, revealing a phase transition in the protein at higher temperatures.
The final part of this thesis describes the application of the ESI-SUPREX methodology
to probe folding properties of CypA in the presence of the self expressed and
purified E. coli chaperonin groEL. Thereby the denaturing properties of groEL have
been emphasised along with the stabilisation of a denatured CypA species.