Development of methodologies for the analysis of glycans by capillary electrophoresis and mass spectrometry

开发毛细管电泳和质谱分析聚糖的方法

• 批准号: RGPIN-2016-03929
• 负责人: Zandberg, Wesley
• 金额: $ 1.82万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=669558
• 关键词: Development; methodologies; analysis; glycans; capillary

Cells make many types of sugars and link them together in diverse combinations to form glycans. Glycans, the most abundant bio-molecules on earth, are an important source of energy and structural materials. Discoveries in the emerging discipline of glycoscience have shown that cells also use glycans to recognize each other and are therefore critical to everything from development to disease. It is the objective of glycoscience to decipher the important biological roles of glycans—these are often facilitated by specific glycan structures. However, the chemical analysis of glycans is a considerable challenge requiring the development of new tools and techniques. The research proposed here seeks to meet this challenge using a combination of instrumental techniques, specifically capillary electrophoresis (CE) and mass spectrometry (MS). I hypothesize that a hyphenated CE-MS technique (a long-term research objective) will be a transformative method of glycan analysis. In support of this objective, new sample preparation and analysis methodologies are required. Glycan analysis by CE requires their prior labelling with fluorescent molecules (fluorophores) to permit sensitive detection, however many of these fluorophores are sub-optimal for MS. Thus new fluorophores suited for both CE and MS will be synthesized and validated. I also propose to develop glycan sample preparation methods that are compatible with these fluorophores. My research will seek to implement these methodologies for the CE and MS analysis of classes of glycans in which the current dearth of robust analytical methods is especially apparent. Specifically, I will focus on three model systems to characterize: (1) the changes in protein-linked glycans during the ripening of cherries, a valuable local (Okanagan) crop; (2) diet-induced changes in gastrointestinal (GI) glycans in mouse models of GI disorders such as colitis; and (3) alterations in pectin, a class of high molecular weight glycan, produced in a collection of flax plants with known mutations in cell wall biosynthetic pathways. In sum, the methods developed through this research program will assist in the efforts of the glycoscience community to structurally characterize glycans; furthermore, the specific systems of proposed study will yield important agriculture, industrial and health benefits. This research will afford students in my lab with excellent training opportunities in modern analytical chemistry, equipping them with research and technical skills that are in high demand in both academia and industry.

细胞制造多种类型的糖，并以不同的组合将它们连接在一起形成多糖。多糖是地球上含量最丰富的生物分子，是能源和结构材料的重要来源。糖科学这一新兴学科的发现表明，细胞也使用多糖相互识别，因此对从发育到疾病的一切都是至关重要的。糖科学的目标是破译多糖的重要生物学作用--这些作用通常由特定的多糖结构促进。然而，多糖的化学分析是一个相当大的挑战，需要开发新的工具和技术。这里提出的研究试图使用仪器技术的组合来应对这一挑战，特别是毛细管电泳法(CE)和质谱仪(MS)。我假设CE-MS联用技术(一个长期的研究目标)将是一种分析多糖的变革性方法。为了支持这一目标，需要新的样品制备和分析方法。用毛细管电泳法分析葡聚糖需要事先用荧光分子(荧光团)标记它们才能进行灵敏的检测，但其中许多荧光团对MS来说并不是最理想的，因此将合成和验证同时适用于CE和MS的新的荧光团。我还建议开发与这些荧光团兼容的葡聚糖样品制备方法。我的研究将寻求将这些方法用于对几类葡聚糖的CE和MS分析，在这些方法中，目前缺乏可靠的分析方法尤其明显。具体地说，我将重点介绍三个模型系统来描述：(1)樱桃成熟过程中蛋白质连接的葡聚糖的变化；(2)饮食诱导的胃肠道(GI)多糖的变化；(3)在结肠炎等胃肠道疾病的小鼠模型中的变化；以及(3)果胶的变化，果胶是一种高分子量的葡聚糖，由一组亚麻植物产生，其细胞壁的生物合成途径已知发生了突变。总而言之，通过这项研究计划开发的方法将有助于糖科学界努力确定多糖的结构特征；此外，拟议的具体研究系统将产生重要的农业、工业和健康益处。这项研究将为我实验室的学生提供极好的现代分析化学培训机会，使他们掌握学术界和工业界都非常需要的研究和技术技能。