2 research outputs found
Electrochemical Reduction of Disulfide-Containing Proteins for Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry
Characterization
of disulfide bond-containing proteins by hydrogen/deuterium
exchange monitored by mass spectrometry (HDX-MS) requires reduction
of the disulfide bonds under acidic and cold conditions, where the
amide hydrogen exchange reaction is quenched (pH 2.5, 0 °C).
The reduction typically requires a high concentration (>200 mM)
of
the chemical reducing agent TrisÂ(2-carboxyethyl)Âphosphine (TCEP) as
its reduction rate constant is decreased at low pH and temperature.
Serious adverse effects on chromatographic and mass spectrometric
performances have been reported when using high concentrations of
TCEP. In the present study, we explore the feasibility of using electrochemical
reduction as a substitute for TCEP in HDX-MS analyses. Our results
demonstrate that efficient disulfide bond reduction is readily achieved
by implementing an electrochemical cell into the HDX-MS workflow.
We also identify some challenges in using electrochemical reduction
in HDX-MS analyses and provide possible conditions to attenuate these
limitations. For example, high salt concentrations hamper disulfide
bond reduction, necessitating additional dilution of the sample with
aqueous acidic solution at quench conditions
Characterizing the Dynamics of α‑Synuclein Oligomers Using Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry
Soluble oligomers formed by α-synuclein
(αSN) are suspected
to play a central role in neuronal cell death during Parkinson’s
disease. While studies have probed the surface structure of these
oligomers, little is known about the backbone dynamics of αSN
when they form soluble oligomers. Using hydrogen/deuterium exchange
monitored by mass spectrometry (HDX-MS), we have analyzed the structural
dynamics of soluble αSN oligomers. The analyzed oligomers were
metastable, slowly dissociating to monomers over a period of 21 days,
after excess monomer had been removed. The C-terminal region of αSN
(residues 94–140) underwent isotopic exchange very rapidly,
demonstrating a highly dynamic region in the oligomeric state. Three
regions (residues 4–17, 39–54, and 70–89) were
strongly protected against isotopic exchange in the oligomers, indicating
the presence of a stable hydrogen-bonded or solvent-shielded structure.
The protected regions were interspersed by two somewhat more dynamic
regions (residues 18–38 and 55–70). In the oligomeric
state, the isotopic exchange pattern of the region of residues 35–95
of αSN corresponded well with previous nuclear magnetic resonance
and electron paramagnetic resonance analyses performed on αSN
fibrils and indicated a possible zipperlike maturation mechanism for
αSN aggregates. We find the protected N-terminus (residues 4–17)
to be of particular interest, as this region has previously been observed
to be highly dynamic for both monomeric and fibrillar αSN. This
region has mainly been described in relation to membrane binding of
αSN, and structuring may be important in relation to disease