基于离子淌度串联质谱技术定量分析蛋白质的空间异构体

Since the conformation of a protein is the primary influencing factor on their function and capacity, such as some isomers might perform different function, it is of great significance to develop a novel method to characterize protein conformation in the accurate, convenient and stable way. Although the analytical strategy for protein conformation based on crystal was widely adopted worldwide, however, it is unavailable for dynamic and on-line quantitative research. By virtue of the unique post-source separation function on structure, ion mobility mass spectrometry is very suitable for identification and quantification study on protein conformation. In this project, ①in order to achieve the native conformation of a protein, the solution and nonvolatile salts would be replaced by dialysis assay, followed by ionized by a chip based ion source; ② After optimization of ion mobility and mass spectrometry parameters, the drift times of protein isomers spent on passing through the drift tube were recorded; ③ Next, the mass spectrum of product and precursor ions would be acquired alternately under data-independent acquisition mode, in addition, a label-free internal standard would be analyzed simultaneously, which could transfer the accurate value to the target protein. Here, the top-down strategy and intact protein would be employed and targeted. After integrating multiply advanced techniques, a novel mass spectrometry method would be developed, which could be applied for protein conformation quantitative research. This method will be an accurate and low-cost way to analysis protein higher structure efficiently. Finally, in the field of quality evaluation and control, application of this method would increase the on-line analysis efficiency and resolve the issue on protein conformational quantification, which will improve the competitiveness of relating products.

蛋白质的空间结构与其功能活性关联紧密，例如，异构体之间的功能往往差异明显。因此，开发精准、便捷、稳定的表征技术有助于深入分析蛋白功能。基于晶体模型的分析策略被广泛用于空间结构的表征中，但其无法开展动态和定量分析。凭借独特的源后结构区分能力，离子淌度串联质谱有望对蛋白空间结构开展定性和定量研究。在本项目中，①为了保护空间结构完整性，采用透析法对蛋白进行溶剂置换和脱盐处理，并导入芯片通道进行离子化；②优化淌度及质谱参数，采集蛋白异构体通过漂移管的迁移时间；③在非数据依赖模式下,交替采集电子传输解离后生成的母、子离子图，通过非标记内标品进行量值传递。项目采用自上而下研究策略，以完整蛋白为目标，综合利用多种质谱技术，建立蛋白空间结构定量分析的质谱新方法，具有高效、准确、成本低的特点。项目的实施有望改善蛋白异构体在线分析效率低、定量困难的问题，可用于蛋白产品的质量评价与控制中，提高产品竞争力。

肽类和蛋白质的功能、活性，与其空间结构密切相关。某些关键位点的氨基酸一旦发生修饰或变化，很可能影响其免疫识别、活性和功能。研究多肽和蛋白质的结构特征、建立高准确度的表征方法，可以对很多生物学、化学研究提供基础数据，对实验结果开展客观解释。基于质谱技术的多肽和蛋白类物质分析方法具有分析速度快、可动态表征的特点，在串联离子淌度谱后，可以在质荷比之外增加额外的离子迁移时间和碰撞横截面积信息，有助于准确鉴定多肽和蛋白类物质的异构体。.本研究基于离子淌度质谱技术，针对多肽和蛋白类物质的结构类似物和异构体，建立了准确鉴定方法，并通过使用非数据依赖质谱采集方式，对心衰诊断标志物B型利钠肽（BNP）中的结构类似物和异构体实现了label-free平行定量。由于采用了氨基酸水解同位素稀释质谱法对内标肽进行了绝对定量，内标肽的质量可以溯源至SI国际单位制，最终，化学合成BNP中的10种主要结构类似肽和异构体定量结果同样也具备了计量学溯源性。.该技术相比于传统方法具有定量结果可溯源、分析效率高的优势，非常适合对多肽和蛋白类物质中的结构类似物进行平行分析。项目围绕多肽结构鉴定、异构体分离分析，开展了大量工作，得益于非数据依赖质谱采集的非歧视效应，以及平行测量能力，该方法具备了更加宽泛的适用度，在多肽和蛋白制品的下游行业中，如制药、体外诊断试剂生产企业，可用于产品的质量控制、杂质分析、工艺改进等环节。