Cross-path reactive chromatography/mass spectrometry as a versatile platform for characterization of primary and higher order structure of complex heterogeneous proteins

交叉路径反应色谱/质谱作为多功能平台，用于表征复杂异质蛋白质的一级和高级结构

• 批准号: 10350609
• 负责人: IGOR A KALTASHOV
• 金额: $ 30.94万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10350609
• 关键词: Affect; Binding; Biological Markers; Characteristics; Charge; Chemicals; Chromatography; Clinical; Complex; Complication; Consumption; Data; Detection; Development; Disulfides; Exclusion; Exhibits; Exposure to; Feedback; Gases; Higher Order Chromatin Structure; Industrialization; Ion Exchange; Ions; Isotope Labeling; Label; Laboratories; Mass Chromatography; Mass Spectrum Analysis; Measurement; Modification; Molecular Conformation; Phase; Polysaccharides; Post-Translational Protein Processing; Process; Production; Protein Analysis; Proteins; Quality Control; Reaction; Reagent; Recombinant Proteins; Research; Sampling; Scheme; Spectrometry, Mass, Electrospray Ionization; Structure; Sulfhydryl Compounds; System; Techniques; Technology; Time; Validation; Variant; base; biophysical properties; disulfide bond; disulfide bond reduction; flexibility; novel; novel strategies; personalized medicine; preservation; protein aggregation; protein complex; protein structure; small molecule; Affect; Binding; Biological Markers; Characteristics; Charge; Chemicals; Chromatography; Clinical; Complex; Complication; Consumption; Data; Detection; Development; Disulfides; Exclusion; Exhibits; Exposure to; Feedback; Gases; Higher Order Chromatin Structure; Industrialization; Ion Exchange; Ions; Isotope Labeling; Label; Laboratories; Mass Chromatography; Mass Spectrum Analysis; Measurement; Modification; Molecular Conformation; Phase; Polysaccharides; Post-Translational Protein Processing; Process; Production; Protein Analysis; Proteins; Quality Control; Reaction; Reagent; Recombinant Proteins; Research; Sampling; Scheme; Spectrometry, Mass, Electrospray Ionization; Structure; Sulfhydryl Compounds; System; Techniques; Technology; Time; Validation; Variant; base; biophysical properties; disulfide bond; disulfide bond reduction; flexibility; novel; novel strategies; personalized medicine; preservation; protein aggregation; protein complex; protein structure; small molecule

PROJECT SUMMARY High-throughput characterization of increasingly complex and heterogeneous protein structures (including both primary and higher order structures) is now required in a variety of fields ranging from personalized medicine (biomarkers) to industrial-scale production of recombinant proteins (for both product quality control and feedback adaptive process control). However, extensive structural characterization usually involves several multi-step processes that are both time- and labor-consuming, and frequently cannot be implemented in a high-throughput format. Additional complication arises from the presence of multiple protein sub-populations in the analytical/clinical/production sample, which may exhibit altered functional or biophysical properties despite having very similar structural characteristics (e.g, small soluble aggregates, aberrant glycoforms, disulfide- scrambled species, etc.). The proposed research aims at developing a robust and versatile analytical technology using the novel cross-path reactive chromatography (XP-RC) platform with on-line detection by electrospray ionization mass spectrometry (ESI MS) augmented by protein ion manipulation in the gas phase (including both conventional top-down MS/MS and the limited charge reduction technique developed in our laboratory). XP-RC allows protein chemical modifications (such as disulfide reduction, covalent labeling, H/D exchange, etc.) to be combined in-line with the separation step and enables real-time MS measurements that are not adversely affected by components incompatible with the ESI process. This is achieved by utilizing the unique elution characteristics (retention) of proteins and small-molecule reagents in non-denaturing chromatographic media (size exclusion or ion exchange); during their retention the proteins can be exposed to various reagents to induce the desired modification(s) in a highly controlled fashion. Our preliminary data provide strong evidence that multiple reactions can be carried out inside a single column in a sequential manner by exposing the protein to multiple reagent plugs as it moves through the column prior to MS detection/characterization of the modified protein. The initial efforts will be focused on implementing differential in-line reduction of disulfide bonds followed by free thiol capping with isotopically labeled reagents for high-throughput disulfide mapping and glycoform profiling (Aim 1). These efforts will be then extended to enable selective reduction of inter-chain disulfides while preserving the non-covalent interactions and internal disulfides in complex protein systems to enable identification of binding partners within such systems; MS/MS detection will allow binding interfaces within such selectively preserved complexes to be localized (Aim 2). An alternative approach will utilize in-line chemical labeling as a means of localizing the binding interfaces. Lastly, the XP-RC/MS platform will be used to implement dilution-free H/D exchange characterization of protein complexes and small soluble aggregates in the top-down fashion (Aim 3).

项目摘要 日益复杂和异质蛋白结构的高通量表征（包括 现在需要在个性化医学的各个领域中进行初级和高级结构） （生物标志物）重组蛋白的工业规模生产（用于产品质量控制和反馈 自适应过程控制）。但是，广泛的结构表征通常涉及多个多步骤 时间和劳动力既耗费且经常在高通量中实施的过程 格式。其他并发症来自于在 分析/临床/生产样本，尽管 具有非常相似的结构特征（例如，小的可溶骨料，异常的糖型，二硫化物 - 炒种类等）。拟议的研究旨在开发强大而多功能的分析技术 使用新型的横向路线反应色谱（XP-RC）平台与电喷雾在线检测 电离质谱法（ESI MS）在气相中通过蛋白离子操纵增强（包括 常规的自上而下的MS/MS和我们实验室开发的有限电荷减少技术）。 XP-RC 允许蛋白质化学修饰（例如二硫键还原，共价标记，H/D交换等）为 与分离步骤结合在一起，并启用并非不利的实时MS测量 受ESI过程不相容的组件影响。这是通过利用独特的洗脱来实现的 在非塑性色谱介质中蛋白质和小分子试剂的特征（保留） （尺寸排除或离子交换）；在保留期间，蛋白质可以暴露于各种试剂以诱导 以高度控制的方式进行了所需的修改。我们的初步数据提供了有力的证据表明 可以通过将蛋白质暴露于 多个试剂塞在MS检测/表征的MS检测/表征之前通过列移动。 蛋白质。最初的努力将集中于实施差异的二硫键在线减少。 通过与同位素标记的试剂的游离硫醇限额，用于高通量二硫化物和糖型 分析（目标1）。然后将扩展这些努力，以使选择性减少链间二硫化物的选择性 保留复杂蛋白质系统中的非共价相互作用和内部二硫化物以实现 在这种系统中识别约束伙伴； MS/MS检测将允许在此类中绑定接口 有选择地保留的要局部的复合物（AIM 2）。另一种方法将使用在线化学 标记作为定位结合界面的一种手段。最后，XP-RC/MS平台将用于实现 自上而下 时尚（目标3）。