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

细胞产生多种类型的糖，并以不同的组合将它们连接在一起形成聚糖。聚糖是地球上最丰富的生物分子，是重要的能源和结构材料来源。糖科学这一新兴学科的发现表明，细胞也使用聚糖来识别彼此，因此对从发育到疾病的一切都至关重要。糖科学的目标是破译聚糖的重要生物学作用，这些作用通常是由特定的聚糖结构促进的。然而，聚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。本文提出的研究旨在利用仪器技术，特别是毛细管电泳（CE）和质谱（MS）的组合来应对这一挑战。我假设连字符CE-MS技术（一个长期的研究目标）将是一种变革性的聚糖分析方法。为了支持这一目标，需要新的样品制备和分析方法。通过CE分析聚糖需要事先用荧光分子（荧光团）标记以允许敏感检测，然而许多这些荧光团对于质谱来说是次优的，因此将合成和验证适合CE和MS的新荧光团。我还建议开发与这些荧光团兼容的聚糖样品制备方法。我的研究将寻求将这些方法用于聚糖类的CE和MS分析，其中目前缺乏可靠的分析方法尤其明显。具体来说，我将重点关注三个模型系统来表征：(1)在樱桃成熟过程中蛋白质连接聚糖的变化，这是一种有价值的当地（奥肯那根）作物；(2)在结肠炎等胃肠道疾病小鼠模型中，饮食引起的胃肠道聚糖变化；(3)在细胞壁生物合成途径中已知突变的亚麻植物中产生的果胶（一类高分子量聚糖）的改变。总之，通过本研究计划开发的方法将有助于糖科学界对聚糖结构表征的努力；此外，拟议研究的具体系统将产生重要的农业、工业和健康效益。这项研究将为我实验室的学生提供现代分析化学方面的良好培训机会，使他们具备学术界和工业界都非常需要的研究和技术技能。

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.