Final Report Summary - A SMILE (analyse Soluble + Membrane complexes with Improved LILBID Experiments)
Non-covalently bound complexes, which can include membrane and soluble proteins as well as nucleotides and ligands, play an essential role in many cellular processes. Mass spectrometry is a well-established method, which can unravel many questions in connection with molecular complexes. A key part of such a mass spectrometer is the ion source, as the transfer of solvated molecules into the gas phase is a critical process, in which the features to be investigated have to be preserved. The traditionally used ion source for non-covalent mass spectrometry is Electro Spray Ionisation (ESI). Despite its very successful use for many applications it is not universally applicable and the development of ion sources with complimentary features can be of great value. Laser Induced Liquid Bead Ion Desorption (LILBID), which is being developed in my lab, is such an alternative method, which is complementary to ESI in many aspects. For LILBID a droplet generator generates on demand, with a frequency of 10Hz, droplets of the analyte solution with 30-50μm diameter. The droplets are then irradiated by an IR-laser, with a wavelength that is tuned to the H-O stretching vibrations of water. This leads to the explosive expansion of the aqueous droplets, setting the solvates biomolecules free. The thus created gas phase ions are comparably low charged and depending on the intensity of the desorption laser the non-covalent bonds between the proteins can be preserved or destroyed. This allows the investigation of the whole complex or its building blocks.
The aSMILE project aims at instrumental improvement, where the home built LILBID instrument is not state of the art as well as the development of a set-up that allows for a LILBID ion source to be coupled to a commercial mass spectrometer.
We optimized several parts of the home-built instrument, including the ion optics, ion transfer and the detector. This minimized peak tailing in the mass spectra and increased the instruments sensitivity. We as well established the relevant aspects which have to be be considered for coupling of a LILBID source to a commercial mass spectrometer, replacing the original ESI source. This involved a different set-up for ion transfer into the instrument as well as modification of instrumental features, where the comparably low charged LILBID ions require different treatment than the higher charged ESI ions.
Additionally we developed new technology, which makes use of the unique aspects of the LILBID method. As the LILBID droplet generators produce analyte droplets on demand they can be used for a defined start of a kinetic reaction. For that means we are developed a droplet Paul trap, which allows storage of slightly charged droplets. The trap allows mixing of two different droplets to start a reaction or uncaging of light sensitive compounds by means of irradiation with a UV laser in the trap. After starting the reaction the droplet can be stored for a defined amount of time. At different time points the droplet can be released from the trap and submitted to the LILBID desorption process for mass analysis. Variation of the storage time will allow to follow the kinetics of fast reactions on time scales from ms to minutes.
We as well developed a means to correlate the amount of desorption laser energy transferred into the droplet, to the degree of dissociation a biomolecular complex undergoes in the desorption process. This allows us to assess binding strength of biomolecular complexes.
In parallel we are working on the analysis of biological samples of interest. This includes for example the screening of potential drugs against Alzheimer’s disease, or the development of handling procedures that allow the investigation of membrane proteins in dependence of the lipids which compose the cell membranes.
The aSMILE project aims at instrumental improvement, where the home built LILBID instrument is not state of the art as well as the development of a set-up that allows for a LILBID ion source to be coupled to a commercial mass spectrometer.
We optimized several parts of the home-built instrument, including the ion optics, ion transfer and the detector. This minimized peak tailing in the mass spectra and increased the instruments sensitivity. We as well established the relevant aspects which have to be be considered for coupling of a LILBID source to a commercial mass spectrometer, replacing the original ESI source. This involved a different set-up for ion transfer into the instrument as well as modification of instrumental features, where the comparably low charged LILBID ions require different treatment than the higher charged ESI ions.
Additionally we developed new technology, which makes use of the unique aspects of the LILBID method. As the LILBID droplet generators produce analyte droplets on demand they can be used for a defined start of a kinetic reaction. For that means we are developed a droplet Paul trap, which allows storage of slightly charged droplets. The trap allows mixing of two different droplets to start a reaction or uncaging of light sensitive compounds by means of irradiation with a UV laser in the trap. After starting the reaction the droplet can be stored for a defined amount of time. At different time points the droplet can be released from the trap and submitted to the LILBID desorption process for mass analysis. Variation of the storage time will allow to follow the kinetics of fast reactions on time scales from ms to minutes.
We as well developed a means to correlate the amount of desorption laser energy transferred into the droplet, to the degree of dissociation a biomolecular complex undergoes in the desorption process. This allows us to assess binding strength of biomolecular complexes.
In parallel we are working on the analysis of biological samples of interest. This includes for example the screening of potential drugs against Alzheimer’s disease, or the development of handling procedures that allow the investigation of membrane proteins in dependence of the lipids which compose the cell membranes.