12 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
Synthetic polypeptides used in the study.
Aggregation of the gelsolin protein fragment is the hallmark of the hereditary systemic disease gelsolin amyloidosis. As with other protein misfolding diseases, there is an urgent need for efficient disease-modifying treatment for gelsolin amyloidosis. The formation of amyloids can be reproduced by incubating the disease-causing amyloidogenic 8 kDa polypeptide, 70-residue gelsolin protein fragment, AGelD187N 173–242, in vitro and monitoring the process by thioflavin T dye. However, for screening of potential aggregation inhibitors, the required protein amounts are large and the biotechnological production of amyloidogenic proteins has many challenges. Conversely, use of shorter synthetic regions of AGelD187N 173–242 does not mimic the in vivo aggregation kinetics of full-length fragment as they have different aggregation propensity. In this study, we present an in vitro aggregation assay for full-length AGelD187N 173–242 that has been produced by solid-phase chemical synthesis and after that monomerized carefully. Chemical synthesis allows us to produce high quantities of full-length fragment efficiently and at low cost. We demonstrate that the generated aggregates are fibrillar in nature and how the purity, terminal modification, initial aggregates and seeding affect the aggregation kinetics of a synthetic gelsolin fragment. We also present sufficient quality criteria for the initial monomerized synthetic polypeptide.</div
Amyloid formation of different synthetic AGelD187N polypeptides.
(A) Amyloid formation kinetics of AGelD187N 173–242 with a 95% purity (red), AGelD187N 173–242 with a 90% purity (purple) and a ThT control (blue) monitored continuously by ThT fluorescence. Three replicate kinetic traces are shown. (B) Amyloid formation kinetics of Ac-AGelD187N 173–243 with a 95% purity (orange) and a ThT control (blue) monitored continuously by ThT fluorescence. Three replicate kinetic traces are shown. (C) Representative electron micrograph of each polypeptide after the aggregation experiment.</p
Electron micrographs of AGelD187N polypeptides after the aggregation experiment.
The original electron micrographs for Fig 2C and one higher magnification electron micrograph of each sample. (PDF)</p
Synthetic AGelD187N 173–242 aggregation assay optimization.
Amyloid formation of synthetic AGelD187N 173–242 in 100 μl reaction volume at (A) 10 μM, pipette seeded with 30 nM seeds, (B) 5 μM, pipette seeded with 30 nM seeds, (C) 10 μM with thread seeding, and (D) 5 μM with thread seeding, monitored continuously by ThT fluorescence. Three replicate kinetic traces are shown.</p
Original gel image for Fig 6A.
Aggregation of the gelsolin protein fragment is the hallmark of the hereditary systemic disease gelsolin amyloidosis. As with other protein misfolding diseases, there is an urgent need for efficient disease-modifying treatment for gelsolin amyloidosis. The formation of amyloids can be reproduced by incubating the disease-causing amyloidogenic 8 kDa polypeptide, 70-residue gelsolin protein fragment, AGelD187N 173–242, in vitro and monitoring the process by thioflavin T dye. However, for screening of potential aggregation inhibitors, the required protein amounts are large and the biotechnological production of amyloidogenic proteins has many challenges. Conversely, use of shorter synthetic regions of AGelD187N 173–242 does not mimic the in vivo aggregation kinetics of full-length fragment as they have different aggregation propensity. In this study, we present an in vitro aggregation assay for full-length AGelD187N 173–242 that has been produced by solid-phase chemical synthesis and after that monomerized carefully. Chemical synthesis allows us to produce high quantities of full-length fragment efficiently and at low cost. We demonstrate that the generated aggregates are fibrillar in nature and how the purity, terminal modification, initial aggregates and seeding affect the aggregation kinetics of a synthetic gelsolin fragment. We also present sufficient quality criteria for the initial monomerized synthetic polypeptide.</div
Lower concentration of synthetic AGelD187N 173–242 provokes sigmoidal aggregation curve and a more complete amyloid formation.
(A) Amyloid formation kinetics of AGelD187N 173–242 at 10 μM (one high-signal and two low-signal reactions in red), at 25 μM (three reactions in purple) and the ThT control (three reactions in blue) monitored continuously by ThT fluorescence. (B) Analytical SEC chromatograms before the aggregation assay (left), after the aggregation assay from the assay supernatant of 25 μM reactions (middle) and from the assay supernatant of the high-signal 10 μM reaction (right).</p
Monomerization yields of different AGelD187N polypeptides.
Monomerization yields of different AGelD187N polypeptides.</p
RP-HPLC and MS data of AGelD187N polypeptides before monomerization.
RP-HPLC and MS data of AGelD187N polypeptides before monomerization.</p
Purity assessment of synthetic AGelD187N 173–242.
Purity assessment of synthetic AGelD187N 173–242 after monomerization (A) on Tricine-SDS-PAGE gel including molecular weight standard (Lane 1) and AGelD187N 173–242 (Lane 2), (B) SEC-MALS analysis and (C) LC-MS analysis; mass spectrum (left) and deconvolved spectrum (right).</p