Brief introduction
I decided to perform my project week in November 2005 in the group of Prof. Abel. The name of the offered project is Analytical laser induced liquid beam desorption mass spectrometry of biomolecules and their aggregates. This topic was especially attractive and interesting for me, as I work in my Ph.D. thesis on a protein, the tau protein, which is supposed to play an important role in Alzheimer’s disease (AD). Tau can be mostly observed in a completely unfolded random-coil state, which resembles a completely unstructured molecular chain. Under certain physiological conditions this protein tends to form aggregates (neurofibrillary tangles), which are one of the most important hallmarks in the course of Alzheimer’s disease. Therefore, elucidation of principles, underlying the aggregating process of tau, is a highly interesting goal, in particular from a medical point of view. The method of mass spectrometry, which is applied in Prof. Abel’s group, should avoid the harshness of other mass spectrometry techniques (like MALDI for example), which causes eventually the degradation of non-covalent aggregates typical for neurodegenerative diseases.

The technical setup
The experimental setup consists of a vacuum chamber pumped by a oil diffusion pump in which a liquid water beam is introduced, a reflectron time-of-flight mass spectrometer pumped by two turbomolecular pumps and an IR Laser System (pulsed Nd:YAG laser, optical parametric oscillator (OPO)). The water beam is generated by a quartz nozzle (~12 µm diameter), the necessary pressure is generated by a HPLC pump at a flow rate of ~0.2 ml/min. An aqueous solution of the sample molecule is introduced into the high-pressure part by a HPLC injection valve. The liquid beam is irradiated by the IR laser tuned to a wavelength of 2.8 µm at the OH-stretch absorption band of water. The laser heats a small volume fraction of the liquid and a shockwave is generated. In the following - not completely understood - process the liquid beam dissolves and ions, mostly singly charged, are formed. The ions drift towards the ion optics where they are collimated and accelerated into the flight tube of the mass spectrometer. After being reflected at the reflectron and passing the second drift region they are 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.

The experiments
Together with Andreas Bögehold (also a member of the Graduate School), I applied this mass-spectrometry technique for investigating the aggregating process of different tau constructs. In humans six different spliceforms of tau can be found. The longest form, htau40, consists of 441 amino acids (molecular mass of ~ 48000 Da). A C-terminal repeat domain in htau40, consisting of 130 residues, forms the core of the AD fibrils and is essential for aggregation. Therefore, we focused on a tau construct, K18, consisting completely of this repeat domain, in addition to htau40. In both cases we worked with the wildtype and with a mutant (DK280) containing a lysine deletion. This mutant should show an enhanced tendency towards aggregation. Furthermore, it has to be mentioned, that polyanions, like heparin, RNA or poly-Glu, also have an accelerating effect on tau aggregation. Thus, heparin can be used as a tool to tune velocity of fibrilization.
We measured three protein solutions:

• 1.) K18-DK280 with a concentration of 4 mg/ml (with heparin (ratio of heparin:tau is 1:4))
• 2.) htau40 wildtype with a concentration of 2 mg/ml
• 3.) htau40-DK280 with a concentration of 3 mg/ml