On-surface glycan sequencing – combining preparative mass spectrometry with scanning probe microscopy to decipher the glycocode

表面聚糖测序 â 将制备型质谱与扫描探针显微镜结合起来破译糖码

• 批准号: 509729094
• 负责人: Marko Grabarics
• 金额: --
• 依托单位: Department of Chemistry
• 依托单位国家: 德国
• 项目类别: WBP Fellowship
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/509729094?language=en
• 关键词: surface; glycan; sequencing; combining; preparative

Biological information and function are encoded in the sequence of biopolymers, such as nucleic acids, proteins, and glycans. Whereas a range of automated, high-throughput methods are available for DNA and protein sequencing, the de novo structure elucidation of glycans is still an unresolved challenge in biochemistry. In contrast to linear biopolymers, glycans tend to form branched structures with complex regio- and stereochemistry, resulting in a vast number of potential isomers. Since isomers have identical mass, conventional tandem MS-based methods often struggle to distinguish them and fail to reveal the unique underlying glycan structure. Thus, there is a clear need for novel analytical techniques that can overcome this limitation and provide all regio- and stereochemical information required for the unambiguous sequence assignment of glycans.The aim of this project is to develop such a de novo glycan sequencing method based on the direct imaging of chemically selected, individual molecules with atomic detail. For this purpose, we will combine preparative MS with state-of-the-art scanning probe microscopy (SPM) techniques under ultrahigh vacuum conditions, and obtain real-space images of mass-selected glycans deposited onto atomically defined surfaces. Employing a unique preparative MS apparatus, we will implement the on-surface glycan sequencing workflow using a set of human milk oligosaccharide (HMO) standards as model compounds. These molecules display all key aspects of glycan structural complexity, providing an ideal platform to develop and optimize the method.Put together, SPM imaging with chemically selective deposition will enable a range of real-world applications that could previously not be tackled. Building on proof-of-concept experiments on HMOs, we will next integrate preparative MS and SPM imaging into established glycomic workflows in order to study protein O-glycosylation. The O-glycan chains released from glycoproteins will be enriched and fractionated by liquid chromatography, and deposited onto clean crystal surfaces by preparative MS to allow for their sequencing using high-resolution SPM.Finally, on-surface glycan sequencing will be extended to study chemical modifications of the carbohydrate backbone. In particular, we will focus on imaging the sulfation pattern of glycosaminoglycans (GAGs), a family of highly acidic polysaccharides. Since the arrangement of sulfate groups governs the binding of bioactive GAG sequences to proteins, deciphering the sulfation code is essential to understanding the structure–function relationships of these molecules. To this end, we will utilize chemically modified SPM probes which, through increasing spatial resolution and chemical selectivity, will facilitate the distinction of functional groups and thus help us determine the position of sulfate modifications in GAGs.

生物信息和功能被编码在生物聚合物的序列中，例如核酸、蛋白质和聚糖。尽管有一系列自动化、高通量的方法可用于DNA和蛋白质测序，但聚糖的从头结构解析仍然是生物化学中一个尚未解决的挑战。与线性生物聚合物相比，聚糖倾向于形成具有复杂区域和立体化学的支链结构，从而产生大量潜在的异构体。由于异构体具有相同的质量，传统的串联MS方法通常难以区分它们，并且无法揭示独特的潜在聚糖结构。因此，有一个明确的需要新的分析技术，可以克服这一限制，并提供所有区域和所需的明确的序列分配的聚糖的立体化学信息。本项目的目的是开发这样一个从头聚糖测序方法的基础上直接成像的化学选择，个别分子与原子的细节。为此，我们将结合联合收割机制备MS与国家的最先进的扫描探针显微镜（SPM）技术在超真空条件下，并获得真实的空间图像的质量选择聚糖沉积到原子定义的表面。采用独特的制备型MS装置，我们将使用一组人乳低聚糖（HMO）标准品作为模型化合物，实施表面聚糖测序工作流程。这些分子显示了聚糖结构复杂性的所有关键方面，为开发和优化该方法提供了理想的平台。总之，SPM成像与化学选择性沉积将实现一系列以前无法解决的现实应用。在HMO概念验证实验的基础上，我们下一步将把制备型MS和SPM成像整合到已建立的糖组学工作流程中，以研究蛋白质O-糖基化。从糖蛋白中释放的O-聚糖链将通过液相色谱法富集和分离，并通过制备型MS沉积在清洁的晶体表面上，以允许使用高分辨率SPM对其进行测序。最后，表面聚糖测序将扩展到研究碳水化合物骨架的化学修饰。特别是，我们将集中在成像的硫酸化模式的糖胺聚糖（GAG），一个家庭的高酸性多糖。由于硫酸基团的排列决定了生物活性GAG序列与蛋白质的结合，因此破译硫酸化密码对于理解这些分子的结构-功能关系至关重要。为此，我们将利用化学修饰的SPM探针，通过增加空间分辨率和化学选择性，将有助于区分官能团，从而帮助我们确定GAG中硫酸盐修饰的位置。