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

与脂质和蛋白质相关的糖基化过程是正常发育和组织内稳态所必需的[1]。研究糖基化对于了解广泛的健康状况很重要，包括伤口愈合。由于多聚糖的复杂性以及在生物环境中研究其空间组织的方法有限，这一领域的发展受到阻碍。飞行时间二次离子质谱仪(ToF-SIMS)可用于化学成像，以前已应用于亚微米分辨率的组织样品[2]。值得注意的是，当SIMS获得每个样本的全谱时，与多糖、脂类和蛋白质相关的信息出现在每个分析中。PI最近展示了ToF-SIMS在纯化糖胺多聚糖(GAGS)的高灵敏度化学识别中的新应用[3]。自2019年CI在诺丁汉推出OrbiSIMS以来，一些研究领域受到了重大影响。其中许多都在生物学研究领域，包括皮肤渗透[4]、干细胞成骨[5]和胶质母细胞瘤内异质性的特征[6]。气体团簇离子束和Orbitrap分析仪相结合，能够产生复杂的、相对较大的生物二次离子，并有能力分别对它们进行准确的分析。迄今为止的顶峰是CI使用SIMS进行从头测序的应用[7]。建议将OrbiSIMS应用于GAG分析也同样有效。在本项目中，OrbiSIMS分析将用于同时对皮肤样本进行血糖和蛋白质组学分析，以了解糖基化在组织稳态和伤口愈合中的作用，朝着确定基于葡聚糖的疗法以帮助伤口愈合的更大目标。WP1-在本WP中，将评估OrbiSIMS在皮肤组织中检测不同糖链的能力。这将包括评估多糖的组成。猪皮将被用作人体组织的模型。通过将感兴趣的生物分子添加到组织匀浆中并使用深度剖析来辅助分析。还将探索飞行时间质量分析仪，以实现更快的分析。将探索各种多变量分析和化学过滤方法来帮助数据解释。WP2-中性损失分析数据的化学过滤将作为从头测序的路线进行研究，以便识别感兴趣的蛋白多糖的特征肽片段。此WP将与WP1并行运行。WP3-项目合作伙伴Hans Wandall(哥本哈根大学)最近开发了皮肤器官型培养，作为一种有吸引力的模型系统，提供了更简单的组织来源，适合高通量基因工程[1]。CRISPR-Cas9基因打靶已被用于建立3D皮肤模型，系统地敲除糖基化中使用的酶。在WP1和2中开发的方法将被应用于这些模型系统，重点是糖基化在伤口愈合中的作用。

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.