Assessing the Biomolecular Structures that Result from Electrospray Ionization

项目摘要/摘要 多糖是碳水化合物的非均相聚合物，与蛋白质相互作用，启动了大量的生物- CAL过程，包括分子识别、细胞信号和宿主-微生物相互作用。质谱仪- 对位计量学(MS)方法已成为表征多糖及其相互作用结构的有力工具。 与蛋白质的作用。电喷雾电离(ESI)是一种常见的电离方法，用于转移这些生物- 分子从溶液到气相进行MS分析。然而，多糖和蛋白质都表现出结构- ESI期间的文化变化。因此，迫切需要了解生物分子结构是如何被修饰的。 在ESI过程中和之后，以确定(1)研究人员如何从这些结构推断出溶剂化结构 技术和(2)如何改进分析方法。 为了应对这一挑战，加拉格尔实验室使用了分子动力学(MD)模拟的组合 和MS方法，以发展ESI的基本分子观点。加拉格尔实验室进行MD 以金属离子为载流子观察碳水化合物电离的ESI模拟。然而，为了更多 为了准确地模拟蛋白质电离，加拉格尔实验室正在开发模拟质子化过程的方法 ESI。这些方法将被进一步发展来研究负离子中去质子化的分析物电离。 模式，然后应用于实现对多糖和蛋白质电离的分子视角。在并列中- 为了进行MD模拟，加拉格尔实验室将对金属加成的葡聚糖进行MS分析。多聚糖 通过与金属离子配位而容易电离，不同的金属加合物使异构体在 串联质谱仪和离子移动质谱仪。然而，多糖通常被分析为钠加合物，因为- 钠是一种无处不在的污染物。加拉格尔实验室正在进行系统的研究，以检验这种关系- 金属离子性质与MS的葡聚糖特性之间的关系。最后，加拉格尔实验室正在应用 天然MS用于表征蛋白质复合体。在原生MS中，非共价相互作用在 气相；然而，过去的工作表明，被分析为正离子和负离子的蛋白质有不同- 对气相稳定性的影响。加拉格尔实验室将研究ESI中的电荷如何形成正极或负极- 阳离子与气相蛋白质的结构、稳定性和配体结合作用有关。 加拉格尔实验室的首要目标是开发和应用新的方法来表征甘氨酸- 罐头、蛋白质及其结合作用。这项提案中描述的研究具有重要意义，因为它 将为ESI-MS提供一个基本的视角，而这项研究的分子洞察力将使 合理开发用于表征这些分子的未来电喷雾质谱方法。最终，这项工作将 便于分析细胞生物学和疾病状态中的多糖、糖偶联物和蛋白质复合体， 使这些方法能够应用于解决重要的生物学假说。

Project Summary / Abstract Glycans, heterogeneous polymers of carbohydrates, interact with proteins to initiate a multitude of biologi- cal processes, including molecular recognition, cellular signaling, and host-microbe interactions. Mass spec- trometry (MS) methods have become powerful tools for characterizing the structures of glycans and their inter- actions with proteins. Electrospray ionization (ESI) is a common ionization method for transferring these bio- molecules from solution to the gas-phase for MS analysis. However, both glycans and proteins exhibit struc- tural changes during ESI. Thus, there is a critical need to understand how biomolecular structures are modified during and after the ESI process to determine (1) how researchers can deduce solvated structures from these techniques and (2) how analytical methods can be improved. To address this challenge, the Gallagher lab uses a combination of molecular dynamics (MD) simulations and MS methods to develop a fundamental, molecular perspective of ESI. The Gallagher lab performs MD simulations of ESI to observe carbohydrate ionization using metal ions as charge carriers. However, to more accurately model protein ionization, the Gallagher lab is developing methods to simulate protonation during ESI. These methods will be further developed to examine analyte ionization by deprotonation in negative-ion mode, and then applied to achieve a molecular perspective on the ionization of glycans and proteins. In paral- lel to the MD simulations, the Gallagher lab will perform MS analysis of metal-adducted glycans. Glycans readily ionize by coordinating to metal ions, with different metal-adducts enabling isomeric differentiation in both tandem MS and ion mobility-MS. However, glycans are often analyzed as sodium adducts because so- dium is a ubiquitous contaminant. The Gallagher lab is performing systematic studies examining the relation- ship between metal-ion properties and glycan characterization by MS. Finally, the Gallagher lab is applying native MS to characterize protein complexes. In native MS, noncovalent interactions are maintained in the gas phase; yet past work has suggested that proteins analyzed as positive versus negative ions have differ- ences in gas-phase stability. The Gallagher lab will examine how charging in ESI to form either positive or neg- ative ions is related to gas-phase protein structure, stability, and ligand-binding interactions. The overarching goal of the Gallagher lab is to develop and apply novel methodologies to characterize gly- cans, proteins, and their binding interactions. The research described in this proposal is significant because it will provide a fundamental perspective on ESI-MS and the molecular insights from this research will enable the rational development of future ESI-MS methods for characterizing these molecules. Ultimately, this work will facilitate the analysis of glycans, glycoconjugates, and protein complexes in cell biology and disease states, enabling these methods to be applied to address important biological hypotheses.