Introduction

Presynaptic protein α-synuclein is a soluble unfolded protein which makes amyloid fibril in vivo and in vitro. The conformational change and kinetic detail from functional monomer to aggregate or amyloid fibril is to be elucidated. While NMR spectroscopy is used to observe atomic level differences in each conformation, liquid beam desorption mass spectrometry is used as complementary method to detect molecular level kinetics in solution.

Sample preparation

C-terminal truncated α-synuclein (residue 1-108; aS108) was expressed by T7 vector in E.coli strain and purified by freeze-and-thaw, sonication, streptomycin sulfate precipitation, ammonium sulfate precipitation and anion-exchange column in sequence. Fibrillization was performed by incubating 200uM aS108 in buffer (20mM Tris, 100mM NaCl at pH 7.5) at 37°C with stirring. At each time point, same amount of the solution was aliquoted, frozen and kept in refrigerator until the measurement.

The experimental setup consist of a vacuum chamber in which a liquid water beam is introduced, a reflectron time-of-flight mass spectrometer and an IR Laser System (pulsed Nd:YAG laser, optical parametric oscillator (OPO)). The water beam was generated by a quartz nozzle (~12 µm diameter) with the necessary pressure by a HPLC pump at a flow rate of ~0.2 ml/min.
An aqueous solution (~ 25uL) of the sample molecule was introduced into the high pressure part by a HPLC injection valve. The liquid beam was irradiated by the IR laser beam tuned to a wavelength of 2.8 µm at the OH-stretch absorption band of water. A small volume fraction of the beam was heated by the laser and a shockwave is generated. In the following - not completely understood process, the liquid beam dissolved and ions, mostly singly charged, were formed. The ions drifted towards the ion optics where they were collimated and accelerated into the flight tube of the mass spectrometer. After being reflected at the reflectron and passing the second drift region they were detected by a micro-channel plate. The spectra closely reflect the conditions in solution and the signal intensities correlate with the solution concentration of the molecule over a wide concentration range.

Exponential decay of monomeric aS108 was observed in series of spectra. As C-terminal is supposed to inhibit aggregation, aS108 aggregates faster and, consequently, monomer signal decrease should be more rapid than full length α-synuclein in the same condition. In approximately 1 day, most of monomer signal depleted.

Figure: Normalized intensity decrease of aS108 monomer signal in solution
(20mM Tris, 100mM NaCl at pH7.5) in a time course.

From this initial measurement, I could find that liquid beam MS is a good method for characterization in macromolecular level and this work will be continued with high resolution mass spectrometer to detect more detailed events including intermediate species.

I heartily appreciate Andreas Boegehold for his skillful help in measurement and data analysis, and Prof. Abel for his support in scientific discussion and instruments.