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Mass Spectrometry 2019
March 04-05, 2019
Berlin, Germany
Mass Spectrometry
9th Edition of International Conference on
International Journal of Drug Development and Research
ISSN: 0975-9344 Page 21
Using native, top-down mass spectrometry to
characterize the interaction of amyloidogenic
proteins with assembly modulator CLR01
Michael Nshanian
University of California, USA
A lzheimer’s disease (AD) is a complex
neurodegenerative disorder that manifests
itself through neuronal death and loss of synaptic
transmission. Its complex pathophysiology includes
a double proteinopathy characterized by aggregation
of the amyloid -protein (A) and neurofibrillary tangles
(NFT) of the microtubule-associated tau protein. Our
group has effectively implemented high resolution mass
spectrometry to study the interaction of amyloidogenic
proteins with lysine specific molecular tweezer (MT)
compound CLR01. CLR01 preferentially binds to Lys
residues on unstructured proteins and modifies their
assembly into non-toxic states. We have employed top
down MS methods to gain structural insight into tau and
tau:CLR01 complexes. In addition, we have discovered
that performing top down ECD MS of noncovalent tau:
CLR01 complex can reveal the site of inhibitor binding.
Noncovalent interactions are generally stable enough
for transition into the gas phase for structural and
stoichiometric analysis. Furthermore, electron capture
dissociation (ECD) based fragmentation preserves
the labile post-translational modifications (PTMs) and
only dissociates the covalent bonds of the noncovalent
complexes,whichisespeciallywellsuitedtoassigningthe
sites of ligand binding. Our top-down MS based methods
have been successfully used to characterize the effects
of CLR01 binding to 4R tau protein (45.8 kDa) and tau
fragment (11 kDa). ESI-MS spectra were obtained for the
unmodified and phosphorylated 4R repeat domain of tau
fragment and tau/CLR01 complex in a 1:1 stoichiometric
ratio. The intact protein-inhibitor complex was further
subjected to ECD-MS to obtain sequence information and
pinpoint the sites of inhibitor binding. ECD-MS data point
to CLR01 binding sites in the microtubule binding region,
implicated in aggregation. Since phosphorylation plays
an important role in tau aggregation, we have also tested
phosphorylated tau to map the sites of phosphorylation.
ECD-MS confirmed phosphorylation at Ser-235. Our ion
mobility experiments on the tau fragment revealed a shift
towards a more compact structure in the presence of
CLR01.
Biography
Michael Nshanian has completed his PhD in Biochemistry and
Molecular Biology at the University of California, Los Angeles,
under the guidance of Professor Joseph Loo. He is currently a
Postdoctoral research fellow at Stanford University School of
Medicine. He has spent several years in the Pharmaceutical in-
dustry in the San Francisco bay area, where he helped develop
and characterize drug candidates using various analytical tech-
niques. Whileworking in the industry, he has also completed an
MS in Chemistry under the guidance of Professor Joseph Pe-
sek. He has published in JACS, Analytical Chemistry, Interna-
tional Journal of Mass Spectrometry and Electrophoresis. His
most recent research on using native mass spectrometry and
ion mobility spectrometry to study protein-inhibitor complexes
will be published in the upcoming issue of the Journal of the
American Society for Mass Spectrometry.
Michael Nshanian, Int J Drug Dev & Res 2019, Volume 11
DOI: 10.21767/0975-9344-C1-005