Glycan Interactions in Health and Disease

碳水化合物(也称为多糖)在许多生物过程中起着关键作用。在过去的20年里，我的实验室开发了最先进的质谱仪(MS)方法。我们应用这些方法来加速发现和表征糖和糖结合蛋白(GBP)之间的生物相关相互作用，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多糖相互作用在人类健康和疾病中所起的作用。在这份Discovery Grant申请中，我们提出了三个新的项目，以突破当前MS在询问多糖结合和处理方面的能力极限。这项研究将首先导致创新的MS方法，以确定新的糖-GBP相互作用，量化它们的热力学性质，并测量生理条件下的CAZyme活性。这些方法还将用于与合作者一起进行的下游研究，以更深入地了解多糖识别和处理的生物学意义，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.建立一种基于MS的鸟枪式糖组分分析方法，用于发现GBP的天然多糖配体。该分析将结合捕捉释放电喷雾电离(CAR-ESI)-MS和时间分辨热解离测量。我们将使用新获得的15T傅里叶变换离子回旋共振质谱仪(加拿大第一台此类仪器)进行分析。然后，我们将应用CAR-ESI-MS来鉴定具有高治疗价值的GBP靶标的天然配体。2.建立了一种基于MS的分析方法，用于同时鉴定和定量GBP与膜中糖脂混合物的结合。该方法的应用将提高Gbps对细胞识别的分子水平的理解。这样的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.建立与生物体液相容的电喷雾质谱(ESI-MS)分析方法，定量检测CAZyme活性。通过与临床医生的合作，我们将把这种分析应用于临床样本，以阐明特定癌症对CAZyme活性的影响。这些见解有可能开发一种新的、非侵入性的方法来诊断各种癌症。计划中的项目每年将培养6名研究生和1名本科生。所有受训人员将获得高性能MS及其在多糖结合和加工中的应用技能。在一些项目中，他们将获得结合和酶分析开发以及免疫学和癌症生物标记物发现方面的专门技能。他们将在为自然科学和工程领域的职业培训方面取得巨大成功的环境中工作，这种环境将出色的研究培训与为每个人的职业目标量身定做的专业发展机会结合在一起。

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.