B6.1: Time-resolved Mass Spectrometry

Laser-Induced-Liquid-Bead-Ion-Desorption (LILBID) is a soft ionisation method for the analysis of biological samples via mass spectrometry (MS). This technique makes it possible to analyse oligonucleotides and proteins, especially membrane proteins, in physiological environment containing different kinds and amounts of buffers, detergents and salts. This technique uses piezo-driven droplet generators to produce small droplets (with a diameter of 50 µm) containing the analyte of interest in physiological conditions. Those droplets are transferred into the vacuum of the MS instrument, where they are irradiated by an IR laser at the absorption wavelength of water at 2.94 µm. The absorption of the IR light leads to an explosive expansion of the droplet whereby the analyte of interest is transferred into the gas-phase. The gas-phase ion can now be analysed by conventional time of flight (TOF) MS.

Time-resolved (TR) MS can be achieved by capturing a sample droplet in a droplet trap and starting a reaction inside the droplet. The droplet can then be stored for a well-defined amount of time and then transferred into the MS instrument for analysis. In the trap the droplet will be levitated to store it for various time delays between reaction start and MS analysis. This can be achieved by charging the droplet slightly and levitate it electrodynamicaly inside a Paul trap. High voltage AC potentials make it possible to hold charged droplets inside a small volume in space. The reaction starting the kinetic inside those levitated droplets can be triggered via uncaging photo-labile groups which sets a well-defined start time. This will allow measurement of time resolved mass spectra of biological samples in a physiological environment.


B6.2: Development and implementation of a LILBID platform for light-triggered kinetics

The PhD student will finish the setup of the droplet trap that allows UV irradiation of a biological sample including a UV switchable reaction component. This will enable time-resolved MS measurements and thus allows investigating kinetic reactions in the timeframe of ms to min. Faster reactions will be lost due to the inherent dead time of the system, while the droplet is transferred from the droplet trap to the desorption chamber, where the dissolved ions are transferred into the gas phase. Further improvements of the droplet trap will allow characterizing even faster kinetics and puts us in the prime position to go deeper into collaborative projects making use of compounds that were developed within the first CLiC grant period.

One of the questions relevant for the understanding of membrane proteins and their function are specifically interacting lipids. Therefore, we need to be able to distinguish between lipids that are just placed in the membrane next to the membrane protein and those that are specifically bound. Several studies using native MS have shown that some lipids remain tightly bound to membrane proteins in the gas phase of the instrument,[X47,48] allowing the detection of the membrane protein lipid complex. The problem here is that specifically bound lipids as well as annular lipids can stay attached to the membrane protein, making it challenging to distinguish. In cases where the membrane protein is submitted in a detergent and lipid containing solution to the MS instrument, bound detergent molecules add to the complexity of the signals. New detergent / lipid molecules that can be removed from the membrane complex upon UV irradiation would solve this problem.