Investigating the contribution of glycans to wound healing using mass spectrometry imaging

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Glycosylation processes associated with both lipids and proteins are required for normal development and tissue homeostasis[1]. Studying glycosylation is important for understanding a broad range of health conditions, including wound healing. Development in this area is hampered by the complexity of glycans and the limited methodologies for studying their spatial organisation within biological environments. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) can be used for chemical imaging and has been previously applied to tissue samples at sub-micron resolution[2]. Notably, as SIMS acquires a full spectrum for each sample, information associated with glycans, lipids and proteins is present in each analysis. The PI has recently demonstrated the novel application of ToF-SIMS for high sensitivity chemical discrimination of purified glycosaminoglycans (GAGs)[3]. Since the launch of the OrbiSIMS in Nottingham by the CI in 2019, a number of areas of research have been significantly impacted. Many of these have been within the biological research space and include skin permeation[4], osteogenesis in stem cells[5] and the characterization of glioblastoma intra-tumor heterogeneity[6]. The combination of the gas cluster ion beams and OrbiTrap analyser enable the generation of complex relatively large biological secondary ions and the capacity to analyse them accurately respectively. The pinnacle to date has been the CI's application to undertake de novo peptide sequencing using SIMS[7]. It is proposed that the application of the OrbiSIMS to GAG analysis could be equally impactful. In this project OrbiSIMS analysis will be used for simultaneous glycomic and proteomic analysis of skin samples in order to understand the role of glycosylation on tissue homoeostasis and wound healing, towards the larger goal of identifying glycan-based therapies to aid wound healing.WP1 - In this WP the ability of OrbiSIMS to spatially detect different glycans within skin tissue will be assessed. This will include assessing glycan composition. Porcine skin will be used as a model of human tissue. Analysis will be aided by spiking biomolecules of interest into tissue homogenates and the use of depth profiling. The time-of-flight mass analyser will also be explored for faster analysis. Various multivariate analysis and chemical filtering approaches will be explored to aid in data interpretation.WP2 - Chemical filtering of the data for neutral loss analysis will be investigated as a route for de novo peptide sequencing, in order to identify peptide fragments that are characteristic of proteoglycans of interest. This WP will run in parallel to WP1. WP3 - Project partner Hans Wandall (University of Copenhagen) has recently developed organotypic cultures of skin as an attractive model system that offer simpler tissue sources that are amenable to high-throughput genetic engineering[1]. CRISPR-Cas9 gene targeting has been used to develop 3D skin models with systematic knockouts of the enzymes used in glycosylation. The methodologies developed in WP1 and 2 will be applied to these model systems, focussing on the role of glycosylation on wound healing.