Novel Methods to Study Metastable Biomolecular Systems with Native LC/MS

在化学系化学测量和成像计划的支持下，马萨诸塞大学阿默斯特分校的伊戈尔·卡尔塔索夫教授和他的团队正在开发强大的分析工具，以研究蛋白质/受体识别和酶反应等过程。这些新工具将在分子水平上促进对复杂生物过程机制的理解，最终促进对这些过程的操纵，以实现预期的结果。因此，新工具不仅可以在化学领域产生重大影响，而且还可以在生物技术和医学领域产生重大影响。研究的内容包括与行业合作者的密切互动，为参与其中的研究生和本科生的教育经验增加了一个有价值的维度。卡尔塔索夫博士和他的团队还在开展外联活动，试图让当地高中生接触到马萨诸塞州大学阿默斯特分校最先进的分析仪器。生物聚合物动力学和结构的特征(单个生物聚合物链的构象和多单元系统的组装)对于更好地理解生化过程既具有挑战性，又很重要。尤其需要能够探测瞬时系统的工具，这些系统在蛋白质聚集、四级结构的组装和进化、与生理伙伴的相互作用以及酶催化和运输现象等过程中遇到。要更好地理解这些现象，需要可靠的动力学数据和伴随转变而来的构象变化知识。为了改进现有的表征能力，Kaltashov团队正在开发一种新的稳健、灵敏和高选择性的实验策略，该策略结合了在自然条件下运行的液相色谱(LC)(例如，尺寸排除色谱)和本地电喷雾电离质谱仪(MS)的在线检测。新的LC/MS平台在气相中结合了离子操作，从而能够研究高度多相体系(例如，蛋白质-聚合物偶联物)。柱上H/D交换和化学还原反应的引入使自上而下的HDX MS/MS能够表征蛋白质的高阶结构和构象动力学。

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Prof. Igor Kaltashov and his group at the University of Massachusetts at Amherst are developing powerful analytical tools to study processes such as protein/receptor recognition and enzymatic reactions. These new tools will advance understanding of the mechanisms of complex biological processes at the molecular level, ultimately facilitating manipulation of these processes to achieve desired outcomes. Thus, the new tools can have significant impact not only in chemistry, but also in biotechnology and medicine. Elements of the research involve close interaction with industrial collaborators, adding a valuable dimension to the educational experience of the graduate and undergraduate students involved. Dr. Kaltashov and his group are also engaging in outreach activities which seek to expose local high school students to the state-of-the-art analytical instrumentation at UMass-Amherst.Characterization of biopolymer dynamics and architecture (conformations of individual biopolymer chains and assemblies of multi-unit systems) is both challenging and important for improved understanding of biochemical processes. There is particular need for tools capable of probing transient systems, which are encountered in processes such as protein aggregation, assembly and evolution of quaternary structures, interaction with physiological partners, and enzyme catalysis and transport phenomena. Improved understanding of these phenomena requires reliable kinetics data and knowledge of conformational changes that accompany transitions. In order to improve on existing characterization capabilities, the Kaltashov group is developing a new robust, sensitive, and highly selective experimental strategy that uses a combination of liquid chromatography (LC) run under native conditions (e.g., size-exclusion chromatography) with on-line detection by native electrospray ionization mass spectrometry (MS). The new LC/MS platform incorporates ion manipulation in the gas phase to enable studies of highly heterogeneous systems (e.g., protein-polymer conjugates). Incorporation of on-column H/D exchange and chemical reduction reactions enables top-down HDX MS/MS characterization of higher order structure and conformational dynamics of proteins.