This contribution reviews our recent investigations involving (bio)molecule desorption and transfer using large argon cluster ions. Experimentally, the interaction of keV Ar cluster ion beams (Ar⁺_1000-5000) with a reservoir of organic molecules was used to transfer (i) peptides and lysozymes onto a collector, with retention of their bioactivity, paving the way to a new solvent-free method for complex biosurface nanofabrication [1,2]; (ii) MALDI matrix molecules on tissues of interest (brain and endometrium cross-sections) in situ the ToF-SIMS instrument for their analysis and imaging with improved sensitivity [3] and (iii) biomolecules from tissues to a series of substrates (including low MW matrix layers), also for an analytical purpose [4]. In parallel, molecular dynamics (MD) simulations provided access to the detailed microscopic view of the interactions, out of reach of the experiments and needed for their understanding. For instance, our reactive force field (ReaxFF) simulations explained the influence of the cluster parameters and surface structure on lysozyme desorption, fragmentation and denaturation [5].

[1] V. Delmez, H. Degand, C. Poleunis, K. Moshkunov, M. Chundak, C. Dupont-Gillain, A. Delcorte, Deposition of Intact and Active Proteins In Vacuo Using Large Argon Cluster Ion Beams, J Phys Chem Lett. 12 (2021) 952–957.

[2] V. Delmez, B. Tomasetti, T. Daphnis, C. Poleunis, C. Lauzin, C. Dupont-Gillain, A. Delcorte, Gas Cluster Ion Beams as a Versatile Soft-Landing Tool for the Controlled Construction of Thin (Bio) Films, ACS Appl Bio Mater. 5 (2022) 3180–3192.

[3] T. Daphnis, B. Tomasetti, V. Delmez, K. Vanvarenberg, V. Préat, C. Thieffry, P. Henriet, C. Dupont-Gillain, A. Delcorte, Improvement of lipid detection in mouse brain and human uterine tissue sections using in situ matrix enhanced secondary ion mass spectrometry, J. Am. Chem. Soc. Mass Spectrom., Submitted.

[4] B. Tomasetti, C. Lauzin, A. Delcorte, Microvolume expansion with large argon clusters: An effective approach to enhance the ion signal in ToF-SIMS, Anal. Chem., Submitted.

[5] S. Bertolini, A. Delcorte, Reactive molecular dynamics simulations of lysozyme desorption under Ar cluster impact, Appl. Surf. Sci. 631 (2023) 157487.

In this work, we demonstrate the feasibility of determining 3D structures of native biomolecular complexes with high resolution and accuracy by integrating 3D-OrbiSIMS with molecular modelling and simulations approaches.

3D-OrbiSIMS combines the GCIB based fragmentation in a SIMS setup with the high speed and resolution of Orbitrap analyzers to facilitate an unprecedented level of mass spectral molecular analysis for a range of materials (hard and soft matter, biological cells and tissues) at pico molar sensitivity. First developed in 2017 for label-free metabolic imaging, it has been successfully applied to a range of fields, including neuroscience, cancer biology, and materials science to study protein localization, lipid composition, and metabolite distribution in situ. However, its application to biolomolecular structure characterisation is yet to be investigated.

We started by optimising our sample preparation and data collection conditions under varying buffer and temperature conditions to ensure that the bimolecular complexes retain their native-like, hydrated, structural integrity. Our results indicate that the 3D-OrbiSIMS spectrum contains more chemical information under cryogenic conditions and the mass spectrum range is sufficient to probe neutral losses of RNA fragments up to 6 nucleotides in length. We further ascertained that this mass range is sufficient to characterise more than 80% of the biologically relevant RNA/P complexes deposited in the PDB. Next, we show that even though the ballistic fragmentation of the samples by the Argon beam is stochastic in nature, the Cryo-OrbiSIMS experiments can reproducibly generate unique mass fingerprints for all bimolecular complexes studied under different physiological conditions and / or biological conditions. Further, peak assignments of the mass spectrum revealed that the mass data also encoded information about the native structures and plasticity of the complexes studied. Finally, by generating novel pipelines for incorporating Cryo-OrbiSIMS data as restraints in molecular modelling algorithms and molecular dynamics simulations, we were able to construct native-like 2D-folds and atomic resolution 3D-structures of the RNA systems studied.

In conclusion, By benchmarking 3D-OrbiSIMS against existing methodologies, we have provided a critical validation of the technique. This positions 3D-OrbiSIMS as a transformative tool for uncovering the intricate details of native biomolecular complexes. Further, we have also successfully pushed the boundaries of technique’s capabilities in order to propel a step-change in structural characterization by native Mass Spectrometry techniques.

Reactive molecular dynamics (MD) simulations were conducted to investigate the desorption and soft landing of proteins transferred into the vacuum using large argon clusters. Experimentally, the interaction of argon cluster ion beams (Ar⁺_1000-5000) with a target biofilm was previously used in such a manner to transfer peptides and lysozymes (with retention of their bioactivity) onto a collector, paving the way to a new solvent-free method for complex biosurface nanofabrication. However, the experiments did not give access to microscopic view of the interactions needed for their full understanding, which can be provided by the MD model.

Our reactive force field (ReaxFF) simulations explain the influence of the cluster parameters (size and kinetic energy) and surface structure (thickness of the protein layer) on the fragmentation and denaturation of the desorbing proteins. In addition, ReaxFF simulations were performed to clarify the soft landing of the lysozymes and their fragments on collectors of different natures (gold and graphite). The results show that fragmentation occurs preferentially during soft landing on the rigid gold surface, but it is also affected by the impact angle of the molecules. Additionally, the presence of defects in the graphite layer can induce reactions between the (fragments of) proteins and the surface.

Secondary Ion Mass Spectrometry (SIMS) is a robust technique for 3D imaging of inorganic, organic, and biological systems, which offers unparalleled sensitivity and spatial resolution. Recently, the introduction of water cluster projectiles dramatically increased the ionization probability enabling for much more detailed molecular composition maps [1, 2].

Our knowledge of the mechanisms involved in the sputtering by water clusters (H₂O)_n is currently limited. The signal enhancement is present only for a very narrow range of kinetic energy per water molecule. Our recent simulations show that this phenomenon is highly correlated with the number of sample molecules ejected with a partial shell of water molecules [3].

Water can also possess a detrimental effect on the sputtering efficiency. In this work, we use the reactive molecular dynamics simulations of a trehalose surface covered with a few-nanometer thin film of water to investigate the effect of the overlayer thickness on the sputtering yield and mass spectrum. We also discuss how dissolving trehalose molecules in the film influences the ejection phenomena.

This work was supported by a Polish National Science Centre Grant 2019/33/B/ST4/01778. MD simulations were performed using the PLGrid infrastructure.

(1) Sheraz née Rabbani, S.; Barber, A.; Fletcher, J. S.; Lockyer, N. P.; Vickerman, J. C. Enhancing Secondary Ion Yields in Time of Flight-Secondary Ion Mass Spectrometry Using Water Cluster Primary Beams. Anal. Chem. 2013, 85 (12), 5654–5658. https://doi.org/10.1021/ac4013732.

(2) Sheraz, S.; Tian, H.; Vickerman, J. C.; Blenkinsopp, P.; Winograd, N.; Cumpson, P. Enhanced Ion Yields Using High Energy Water Cluster Beams for Secondary Ion Mass Spectrometry Analysis and Imaging. Anal. Chem. 2019, 91 (14), 9058–9068. https://doi.org/10.1021/acs.analchem.9b01390.

(3) Kański, M.; Hrabar, S.; van Duin, A. C. T.; Postawa, Z. Development of a Charge-Implicit ReaxFF for C/H/O Systems. J. Phys. Chem. Lett. 2022, 13 (2), 628–633. https://doi.org/10.1021/acs.jpclett.1c03867.