Glycan Interactions in Health and Disease

碳水化合物（也称为聚糖）在许多生物过程中发挥着关键作用。在过去的 20 年里，我的实验室开发了最先进的质谱 (MS) 方法。我们应用这些方法来加速聚糖和聚糖结合蛋白（GBP）之间的生物学相关相互作用的发现和表征，并评估碳水化合物活性酶（CAZymes）的活性和底物特异性。通过这些方法，我们进一步了解了这些聚糖相互作用在人类健康和疾病中发挥的作用。在本次发现资助申请中，我们提出了三个新项目，这些项目突破了当前 MS 检测聚糖结合和加工能力的极限。这项研究首先将导致创新的 MS 方法来识别新的聚糖-GBP 相互作用、量化其热力学性质并测量生理条件下的 CAZyme 活性。这些方法还将与合作者一起用于下游研究，以更深入地了解聚糖识别和加工的生物学意义，并将这些发现转化为新的治疗和诊断方法。我们的三个项目是： 1. 开发基于 MS 的鸟枪糖组学测定，以发现 GBP 的天然聚糖配体。该测定将结合捕获和释放电喷雾电离 (CaR-ESI)-MS 和时间分辨热解离测量。我们将使用新购置的 15T 傅里叶变换离子回旋共振质谱仪（加拿大第一台）来实施该测定。然后我们将应用CaR-ESI-MS来鉴定具有高治疗价值的GBP靶点的天然配体。 2. 开发基于 MS 的检测方法，以同时识别和定量 GBP 与膜中糖脂混合物的结合。该测定的应用将提高对 GBP 细胞识别的分子水平理解。这些见解将指导针对糖脂的抑制剂和治疗性抗体的合理设计。 3. 开发与生物液体兼容的 ESI-MS 检测方法，以量化 CAZyme 活性。通过与临床医生合作，我们将将该检测应用于临床样本，以阐明 CAZyme 活性如何受到特定癌症的影响。这些见解有可能开发出一种新的非侵入性方法来诊断各种癌症。计划项目每年将培养六名研究生和一名本科生。所有学员都将获得高性能 MS 及其在聚糖结合和加工中的应用的技能。在一些项目中，他们将获得结合和酶测定开发以及免疫学和癌症生物标志物发现方面的专业技能。他们将在一个非常成功地培训个人从事自然科学和工程职业的环境中工作，该环境将出色的研究培训与针对每个人的职业目标量身定制的专业发展机会相结合。

Carbohydrates (also called glycans) play critical roles in many biological processes. Over the past 20 years my lab has developed state-of-the-art mass spectrometry (MS) methods. We apply these methods to accelerate discovery and characterization of biologically-relevant interactions between glycans and glycan-binding proteins (GBPs) and evaluate the activities and substrate specificities of Carbohydrate-Active enZymes (CAZymes). With these methods, we further our understanding of the roles that these glycan interactions play in human health and disease. In this Discovery Grant application, we propose three new projects that push the limits of current MS capabilities for interrogating glycan binding and processing. This research will lead first and foremost to innovative MS methods to identify new glycan-GBP interactions, quantify their thermodynamic properties and measure CAZyme activities under physiological conditions. These methods will also be used in down-stream studies with collaborators to build a deeper appreciation of the biological implications of glycan recognition and processing and to translate these findings into new therapeutics and diagnostics. Our three projects are: 1. Developing a MS-based shotgun glycomics assay for discovery of natural glycan ligands of GBPs. The assay will combine catch-and-release electrospray ionization (CaR-ESI)-MS and time-resolved thermal dissociation measurements. We will implement the assay using a newly acquired 15T Fourier transform ion cyclotron resonance mass spectrometer (the first of its kind in Canada). We will then apply CaR-ESI-MS to identify natural ligands of GBP targets that have high therapeutic value. 2. Developing a MS-based assay to simultaneously identify and quantify GBP binding to mixtures of glycolipids in membranes. Application of this assay will improve molecular-level understanding of cell recognition by GBPs. Such insights will guide the rational design of inhibitors and therapeutic antibodies that target glycolipids. 3. Developing an ESI-MS assay that is compatible with biological fluids, to quantify CAZyme activities. Through collaborations with clinicians we will apply the assay to clinical samples to elucidate how CAZyme activities are affected by specific cancers. These insights have potential in developing a new, non-invasive method to diagnose various cancers. The planned projects will train six graduate students and one undergraduate each year. All trainees will gain skills in high-performance MS and its applications to glycan binding and processing. In some projects, they will gain specialized skills in binding and enzyme assay development, and in immunology and cancer biomarker discovery. They will work in an environment that has been highly successful in training individuals for careers in the natural sciences and engineering, one that combines outstanding research training with professional development opportunities tailored to each individual's career objectives.