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

项目总结 高通量表征日益复杂和异质的蛋白质结构(包括两者 初级和更高级结构)现在需要用于从个性化医疗的各种领域 (生物标志物)到工业规模的重组蛋白生产(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 既耗时又耗力，而且经常不能以高吞吐量实施的流程 格式化。更复杂的情况是由于存在多个蛋白质亚群 分析/临床/生产样本，可能表现出改变的功能或生物物理性质，尽管 具有非常相似的结构特征(例如，小的可溶聚集体，反常的糖形式，二硫化物- 杂乱的物种等)。拟议的研究旨在开发一种健壮和通用的分析技术 采用新型跨路电喷雾在线检测的XP-RC平台 气相中蛋白质离子操作增强的电离质谱学(包括两者 传统的自上而下的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(如二硫键还原、共价标记、H/D交换等)成为 与分离步骤相结合，实现实时MS测量，而不会对 受与ESI流程不兼容的组件影响。这是通过使用独特的洗脱来实现的 蛋白质和小分子试剂在非变性层析介质中的特性(保留) (大小排斥或离子交换)；在保留期间，蛋白质可暴露于各种试剂以诱导 想要的修改(S)以高度受控的方式。我们的初步数据提供了强有力的证据 通过将蛋白质暴露在一个柱子内，可以连续地进行多个反应 在MS检测/表征改性前穿过柱子时的多个试剂塞 蛋白。最初的努力将集中在实施差异化的二硫键在线还原上，然后是 通过用同位素标记试剂覆盖游离硫醇来高通量地绘制二硫键图谱和糖形 剖析(目标1)。然后，这些努力将扩展到能够选择性地还原链间二硫化物，同时 保留复杂蛋白质系统中的非共价相互作用和内部二硫键，以使 识别此类系统内的绑定伙伴；MS/MS检测将允许在此类系统内绑定接口 选择性地保留要定位的复合体(目标2)。另一种方法是使用在线化学品 标记作为本地化绑定接口的一种方法。最后，使用XP-RC/MS平台来实现 自上而下蛋白质复合体和小聚集体的无稀释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).