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

项目摘要 高通量表征日益复杂和异质的蛋白质结构（包括 初级和高级结构）现在在从个性化医学 （生物标志物）到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）。然而，广泛的结构表征通常涉及几个多步骤 这些过程既耗时又耗力，而且经常无法以高吞吐量的方式实现。 格式.另外的复杂性是由于蛋白质组中存在多个蛋白质亚群引起的。 分析/临床/生产样品，尽管 具有非常相似的结构特征（例如小的可溶性聚集体、异常糖型、二硫化物- 乱序物种等）。拟议的研究旨在开发一种强大而通用的分析技术 使用新型交叉路径反应色谱（XP-RC）平台，通过电喷雾进行在线检测 通过气相中的蛋白质离子操作增强的电离质谱（ESI MS）（包括 常规的自顶向下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（如二硫键还原、共价标记、H/D交换等）是 与分离步骤在线组合，并能够进行实时MS测量， 受与ESI工艺不兼容的组件影响。这是通过利用独特的洗脱 蛋白质和小分子试剂在非变性色谱介质中的特性（保留） (size排斥或离子交换）;在其保留期间，蛋白质可暴露于各种试剂以诱导 以高度受控的方式进行所需的修改。我们的初步数据提供了强有力的证据，表明 通过将蛋白质暴露于 在MS检测/表征改性的前体之前，当其移动通过柱时， 蛋白最初的努力将集中在实施二硫键的差分在线还原， 通过用同位素标记的试剂进行游离巯基加帽，用于高通量二硫键图谱和糖型 剖析（目标1）。然后，这些努力将扩展到能够选择性还原链间二硫化物， 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， MS/MS检测将允许这种系统内的结合界面 选择性地保存待定位的复合物（目的2）。另一种方法将利用在线化学品 标记作为定位结合界面的手段。最后，将使用XP-RC/MS平台来实现 自上而下的蛋白质复合物和小的可溶性聚集体的免稀释H/D交换表征 时尚（目标3）。

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).