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Mass Spectrometry for Single Particle Imaging of Dipole Oriented protein Complexes

Periodic Reporting for period 3 - MS SPIDOC (Mass Spectrometry for Single Particle Imaging of Dipole Oriented protein Complexes)

Reporting period: 2022-06-01 to 2023-06-30

The European XFEL with its unparalleled peak brilliance and repetition rate has the potential to further applications in single particle imaging (SPI), thus far limited to large viral particles at X-ray Free-Electron Lasers (XFEL). SPI will allow imaging protein complexes without the need for crystallization. This eventually renders transient conformational states accessible for high resolution structural studies yielding molecular movies of biomolecular machines. A major bottleneck is the wealth of data required to reconstruct a single structure leading to long processing times.
MS SPIDOC will overcome this data challenge by developing a native mass spectrometry (MS) system for sample delivery, named X-MS-I. It will provide mass and conformation selected biomolecules, which are oriented along their dipole axis upon imaging. This will enable structural reconstructions from much smaller datasets speeding up the analysis time tremendously. Moreover, the system features low sample consumption and a controlled low background easing pattern identification.
The main objectives of the project are:
• Deliver mass and conformation separated biomolecules for SPI.
• Orient proteins for SPI.
• Image protein complex unfolding.
• Exploit potential of protein orientation for other applications.
The MS SPIDOC consortium combines internationally leading expertise in different fields relevant to the project.
All components of the prototype were delivered to EuXFEL after initial design, testing and further optimization. The ITI is fully tested, data will be prepared for publication. The DIT has been tested in depth at UG and the results have been published. The IM has passed initial tests and more in-depth tests are ongoing. It has also been employed in first synchrotron experiments. The overall prototype has been assembled and different versions/iterations of it used in synchrotron experiments. All components as well as the prototype matched or exceeded the design specifications.
For the integration of X-MS-I at SPB/SFX, feasibility simulations for SPI of norovirus-like particles (T=1, T=3) have been performed and published. To flange X-MS-I to the SPB/SFX instrument chamber, a stand for the prototype was designed, constructed and delivered. The prototype itself is coupled to a five-dimensional motion system (X, Y, Z, tip, tilt) for the experiment. X-ray diffractive imaging experiments with the X-MS-I prototype have been performed at the PETRA IIII synchrotron. Beamline integration was successful; however, beamline background dominated the scattering data.

A light version of the prototype (lacking dipole finger and ToF) has been assembled and tested. We have looked into the adaptation of the X-MS-I prototype for the SQS-instrument at the European XFEL to extend its experimental capabilities.
We have designed and build an ESA-ToF as the available footprint was insufficient for a regular ToF providing the desired resolution and mass range.

With regard to the implementation of spectroscopic approaches on the prototype, the practicalities of laser coupling have been examined. There, the ion residence time prevents the use of a CW (continuous wave) laser due to limiting peak power. As a result, a pulsed ns laser was used to achieve sufficient efficiency. Theoretical assessment of collision induced unfolding of proteins and comparison to experimental unfolding using action spectroscopy of labelled ubiquitin mutants has been performed.
To investigate the conformational stability of activated proteins, we have also emulated collision induced unfolding (CIU) in MD simulations by using elevated temperature as a proxy for the activating collisions. The results have been submitted for publication and the manuscript is currently being revised with experimental IM data.

We investigated the impact of rehydration on gas phase protein structures by extensive MD simulations to rehydrate and relax proteins that were previously exposed to vacuum and electric fields. The published results indicate that the effect of electric fields on protein structures becomes less significant after rehydration compared to the impact of vacuum exposure and ionization during electrospraying.

To study protein orientation, Fasmatech has designed and simulated a compact accelerator system capable of sustaining a strong electric field in the range of 25-30 kV/mm. This device uses the intrinsic dipole moment of protein complexes to allow a defined imaging angle along one axis to enhance diffraction pattern alignment. A first prototype of the so-called high voltage finger has been designed, constructed and tested successfully in Fasmatech’s R&D lab. A static electric field intensity of 25 kV/mm has been accomplished without significant effort in surface finish. Finally, a series of simulations have been performed and ion residence time in the interaction zone has been calculated and compared to the time frame of ion orientation predicted by MD simulations. An orientation device with surrounding optics has been designed and delivered to EuXFEL achieving fields up to 50 kV/mm.
Within the whole duration of the project the major aim of developing and assembling the functional prototype has been achieved. However, imaging experiments at EuXFEL have not yet been performed due to lack of beamtime. The developed and tested components show good functionality with respect to required specifications. Moreover, UU and HPI/LIV will exploit dipole orientation for gas phase SAXS in the near future. The overall expected impact as well as an update on identified risks is presented below.
The knowledge and expertise acquired during the project will be further developed in the newly funded SPIDoc’s doctoral network. This also includes imaging experiments, for which several proposals have been submitted to EuXFEL.
The obtained projects’ results and technology developments have been published and new publications are in preparation. The design of components of the X-MS-I prototype was optimized for their functionality for the X-MS-I prototype which has been assembled and tested, as reported. The optimization of components will be exploited for marketing potential.