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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

基本信息

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
项目类别：
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

项目摘要

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非变性色谱介质中蛋白质和小分子试剂的特征（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，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）。