Enabling Ion Mobility Mass Spectrometry for Glycomics

在后基因组时代，生物学比几年前想象的要复杂得多，这一点变得越来越明显。在发现非编码遗传物质和DNA修饰彻底改变了我们对基因组学的看法的同时，基因产物修饰的无处不在的存在正迫使我们重新评估这些蛋白质的复杂性。糖与蛋白质的结合(糖基化)是所有蛋白质修饰中最普遍的。这些所谓的多糖影响蛋白质的功能、结合其他生物分子的能力以及蛋白质在细胞内外的运输。此外，糖链结构的变化与多种疾病有关，而这些糖分子是有趣的生物标记物。然而，尽管它们非常重要，但由于其结构的复杂性，识别和分配多糖仍然是一个巨大的挑战。葡聚糖通常由10-15个单糖残基组成，排列成错综复杂的分支结构。由于它们在细胞中的组装不是模板驱动的，因此多糖是由糖构建块组成的令人难以置信的多样化组装，使得它们的结构阐明非常困难。对给定的多糖进行表征通常需要多种分析方法的结合，这些方法围绕着高效液相色谱(HPLC)和质谱仪(MS)的基石构建。目前这些实验的策略在15年内基本上没有变化，在这段时间里，MS仪器的显著改进对我们表征多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很复杂，需要相当长的时间和专业知识才能解释。我们建议利用一种新型的MS方法--离子迁移率MS(IM-MS)的出现来开发一种现代糖科学的新工具。IM是一种独特的技术，可以在惰性气体中根据大小和形状分离不同的多糖结构。我们在此提供原则证明数据，以证明IM在糖组学中的潜在用途，并为其应用制定一种新的工具。这将被用来实现结构的分离，并通过测量它们的碰撞截面(CCS)来提高指定未知多糖的置信度。我们的建议是基于我们对葡聚糖离子如何在气相中迁移的探索性研究。我们已经证明，IM对多糖的分离和分配严重依赖于特定金属离子加合物的存在。这使我们能够描述一种策略，通过该策略，我们将测量来自合成的和常见的生物衍生结构的糖离子和加合物的CCS值。重要的是，当前商业上可用的IM-MS仪器不能绝对测量CCS，因此在分析之前需要校准。测量将在牛津独特的仪器上进行，该仪器能够确定CCS值，而不需要校准。这些绝对值将通过我们的数据库GlycoMob提供给社区，提供合适的校准数据集和将IM整合到LC-MS糖组分管道中的框架。最后，我们将把IM-MS与高效液相色谱和气相裂解相结合，以实现无与伦比的分析水平，为每个样本赋予特定糖链的四个特征信息片段。我们期待这个工具能够彻底改变我们发现糖组分的方法，以及它所支持的不同研究领域。

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.