Enabling Ion Mobility Mass Spectrometry for Glycomics

在后基因组时代，越来越明显的是，生物学比几年前想象的要复杂得多。在发现非编码遗传物质和DNA修饰的同时，基因产物修饰的普遍存在也迫使我们重新评估这些蛋白质的复杂性。糖与蛋白质的连接（糖基化）是所有蛋白质修饰中最普遍的。这些所谓的聚糖影响蛋白质功能，结合其他生物分子的能力以及细胞内外的蛋白质运输。此外，聚糖结构的变化与多种疾病相关，这些糖分子是作为生物标志物的有趣靶点。然而，尽管它们非常重要，但由于其结构的复杂性，聚糖的鉴定和分配仍然是一个巨大的挑战。聚糖通常由10-15个单糖残基组成，排列为复杂的分支结构。由于它们在细胞中的组装不是模板驱动的，聚糖是糖构建块的难以置信的多样化组装，使得它们的结构解析非常困难。给定聚糖的表征通常需要结合围绕高效液相色谱法（HPLC）和质谱法（MS）的基础构建的多种分析方法。这些实验的当前策略在>15年内基本上没有变化，在此期间MS仪器的显著改进对我们表征聚糖结构的能力几乎没有影响。此外，从这些实验中获得的数据往往是复杂的，需要大量的时间和专业知识来解释。我们建议利用一种新型的MS方法，离子迁移率MS（IM-MS）的出现，为现代糖科学开发一种新的工具。IM是一种独特的技术，可以在惰性气体中通过大小和形状分离不同的聚糖结构。我们在这里提出的原理证明的数据，以证明糖组学的IM的潜在效用，并制定了一个新的工具，其应用。这将用于分离结构，并通过测量其碰撞截面（CCS），提高未知聚糖分配的置信度。我们的建议是建立在我们的探索性研究，了解如何聚糖离子迁移在气相中。我们已经表明，分离，因此分配，聚糖IM是严重依赖于特定的金属离子加合物的存在。这使我们能够描绘一个战略，我们将测量CCS值的聚糖离子和加合物从合成和常见的生物衍生的structure.Importantly，目前市售的IM-MS仪器是不能绝对CCS测量，因此需要在分析之前进行校准。测量将在牛津的独特仪器上进行，该仪器能够在不需要校准的情况下确定CCS值。这些绝对值将通过我们的数据库GlycoMob提供给社区，该数据库提供了合适的校准数据集和将IM整合到LC-MS糖组学管道中的框架。最后，我们将IM-MS与HPLC和气相裂解相结合，以实现无与伦比的分析水平，为每个样品集提供四条特征信息，以“指纹”特定聚糖。我们预计，这一工具可以彻底改变我们的方法，糖组学的发现，以及它所支持的不同领域的研究。

In the post-genomic era it has become increasingly apparent that biology is far more complicated to what was imagined just a few years ago. In parallel to the discovery of non-coding genetic material and DNA modifications that have revolutionised our view of genomics, the ubiquity of gene-product modifications is necessitating us to reassess the complexity of these proteins. The attachment of sugars to proteins (glycosylation) is the most prevalent of all protein modifications. These so-called glycans influence protein function, ability to binds other biomolecules as well as protein transport within and outside the cell. Furthermore, changes in glycan structure are associated with a wide range of diseases, and these sugar molecules are interesting targets as biomarkers. Despite their great importance however, the identification and assignment of glycans remains a great challenge, due to the complexity of their structures. Glycans are typically composed of 10-15 monosaccharide residues, arranged as intricate branched structures. As their assembly in the cell is not a template driven, glycans are incredibly diverse assemblies of sugar building blocks, rendering their structural elucidation very difficult. Characterization of a given glycan generally requires the combination of multiple analytical approaches built around the cornerstones of high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Current strategies for these experiments are essentially unchanged in >15 years, with the significant improvements in MS instrumentation over that time having had little impact on our ability to characterize glycan structures. Furthermore, the data obtained from these experiments is often complicated and requires considerable time and expertise for its interpretation. We propose to capitalise on the advent of a new type of MS approach, ion mobility MS (IM-MS) to develop a new tool for modern glycoscience. IM is a unique technology that can separate different glycan structures by their size and shape in an inert gas. We present here proof-of-principle data to demonstrate the potential utility of IM for glycomics, and formulate a new tool for its application. This will be used to impart separation of structures, and through measuring their collision cross-sections (CCS), improve confidence in assignment of unknown glycans. Our proposal is founded on our exploratory studies into understanding how glycan ions migrate in the gas phase. We have shown that the separation, and therefore assignment, of glycans by IM is critically dependent on the presence of specific metal ion adducts. This enables us to delineate a strategy whereby we will measure CCS values of glycan ions and adducts from synthetically and common biologically derived structures.Importantly, current commercially available IM-MS instrumentation is not capable of absolute CCS measurements and therefore requires calibration prior to analysis. Measurements will be performed on unique instrumentation in Oxford that is able to determine CCS values without the need for calibration. These absolute values will be made available to the community through our database GlycoMob providing both a suitable calibrant dataset and a framework for integrating IM into the LC-MS glycomics pipeline. Finally we will couple IM-MS to HPLC and gas-phase fragmentation for unsurpassed levels of analysis, imparting four characteristic pieces of information for each sample set to 'fingerprint' specific glycans. We anticipate that this tool could revolutionise our approach to glycomics discovery, and the diverse fields of research it underpins.