Glycosaminoglycans (GAGs) are important biopolymers situated within the peri-cellular and extra-cellular spaces that have multiple biological roles. They are complex molecules with variations in sulphation, acetylation and epimerisation that results in different biological behaviour.

Secondary ion mass spectrometry (SIMS) has increasingly been used for analysing biological samples, with a wealth of studies on DNA and proteins but limited analysis of GAGs. In this presentation I will present my recent endeavours to address this gap, making use of gene-editing tools to create robust biological reference samples and high mass resolution Orbi-SIMS analysis to enable identification of GAG-derived ions. I will demonstrate the utility of SIMS as an effective tool to probe the complexity of GAGs within biological samples.

With the increasing global demand for food, the need to produce newer crop-protection products is also growing for efficient and productive farming. For the commercial production of agrochemicals, it is pivotal to understand their distribution and metabolism in the plant system. The method currently used to understand this is autoradiography, a tedious technique usually done at the far end of product development. In this study, we aim to develop more convenient, alternate methodologies for imaging xenobiotics with mass spectrometry.¹ The main bottleneck to doing so is that there are no well-established protocols for sample preparation and application of xenobiotics to study localization in plants compatible with mass spectrometry imaging.² Hence, we are trying to identify suitable mass spectrometry imaging technologies including SIMS, DESI and MALDI for analysis and the corresponding sample preparation taking Azoxystrobin as a model fungicide along with wheat and tomato plants as model plants.

The main aim of this study is to visualize the localization of xenobiotics once applied to the leaf across the leaf lamina and veins and into the depths of the leaf. Initially, the analysis parameters for Azoxystrobin formulation were identified for different MSI techniques. To understand the surface localization of xenobiotics once absorbed into the leaf, the direct analysis of the leaf surface is futile. Hence, some surface modifications like chloroform dipping of the leaf or indirect analysis by forceful leaf imprinting onto another porous surface need to be done. Complimentary to this, cryo-embedding and cryo-sectioning of leaves could be done to understand the depth penetration of the applied xenobiotics. We explored the possibilities of direct and indirect analysis of leaves with different materials and found porous PTFE to be one of the best possible options to understand surface localizations. Since leaf tissues are quite fragile, for sectioning, we also explored the best embedding conditions for them like appropriate embedding media and pre- and post-sectioning methods to produce the best sections possible without chemical delocalization. For sectioning, we also explored the use of specialized mass spectrometer-compatible tapes to reduce sample damage while sectioning.

Such sample preparation and analysis methodologies could be used for understanding the time-dependent localization of agrochemicals when applied to the plant leaves and could act as a complementary technique to the existing visualization methodologies to produce safer agrochemicals.

References:

1. Lorensen, M. D. B. B., Bjarnholt, N., St-Pierre, B., Heinicke, S., Courdavault, V., O'Connor, S., & Janfelt, C. (2023). Spatial localization of monoterpenoid indole alkaloids in Rauvolfia tetraphylla by high resolution mass spectrometry imaging. Phytochemistry, 209, 113620.

1. Ajith, A., Milnes, P. J., Johnson, G. N., & Lockyer, N. P. (2022). Mass Spectrometry Imaging for Spatial Chemical Profiling of Vegetative Parts of Plants. Plants, 11(9), 1234.

The worldwide experience with the COVID 19 pandemic demonstrated once more that without the prevention and the development of specific pharmaceutical products the reduction of the risk of infection can be achieved only by tracing positive cases while imposing strong restrictions to the population. However, this type of measures affected dramatically the economy and the social activities making the research to find a vaccine very urgent and necessary.

The development of vaccine is not an easy task especially when pure antigens are employed for reducing vaccine immunogenicity. This requires the use of adjuvants to optimize vaccine effects whilst maintaining its safety.

Adjuvants based on Aluminium salts are amongst the most used because of its high safety profile, whilst its mechanisms of actions remain unclear.

In this work, we present a detailed surface analysis by means of Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and X-ray Photoemission Spectroscopy (XPS of a polymeric platform to be used for vaccine development and delivery. This platform consisted in Al containing polymer substrate prepared in two different formulations, namely micro-Alum and nano-Alum. The effectiveness of the Alum-platform was tested against ovalbumin (OVA) protein.

Both XPS and ToF-SIMS were able to deliver information about the interaction of the ovalbumin and the substrate. In particular, results indicate that, independently of the substrate formulation (micro or nano), a ratio of Alum/OVA of 3:1 was required to reach saturation. These results were confirmed by SDS-PAGE gel measurements where the 3:1 ratio was the first supernatant sample in which OVA protein could be detected. Moreover, circular dichroism analysis on Al:OVA complexes shown a conformational change of the protein secondary structure upon adjuvant binding.

Unraveling characteristic compositional patterns of extracellular matrices (ECM) promises new insights into mechanistic biology, unprecedented bioengineering options, and progress in medical diagnostics. Analytical techniques to reliably identify ECM variants are therefore of high and growing interest. Since many standard approaches (immunostaining, enzyme-linked immunosorbent assays, Western blotting) applied for that purpose require prior knowledge of the sample and/or tedious sample processing, mass spectrometry approaches are increasingly considered a valuable and widely explored alternative. Our study [1] for the first time demonstrates the potential of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to detect subtle differences between human mesenchymal stromal cell (MSC)-secreted matrices as induced by exogenous stimulation or emerging pathology. For that aim, ToF-SIMS spectra of decellularized ECM samples were evaluated by discriminant principal component analysis (DPCA), an advanced multivariate analysis technique, to decipher characteristic compositional features. To establish the approach, signatures of major ECM proteins were determined from samples of pre-defined mixtures. Based on that, sets of ECM variants produced by MSCs in vitro were analyzed. Differences in the content of collagen, fibronectin, and laminin in the ECM resulting from the combined supplementation of MSC cultures with ascorbic acid and macromolecular crowding agents could be detected by DPCA of ToF-SIMS spectra. The results were verified by immunostaining. Finally, the comparative ToF-SIMS analysis of ECM produced by MSCs of healthy donors and patients suffering from myelodysplastic syndrome (MDS), a disease that arises from genetically altered hematopoietic stem cells in the bone marrow, displayed the potential of the methodology to reveal disease-associated alterations of the ECM composition.

[1] R. Zimmermann, M. Nitschke, V. Magno, U. Freudenberg, K. Sockel, F. Stölzel, M. Wobus, U. Platzbecker, C. Werner, Small Methods 2023, 2201157